Genetically modified tumor infilitrating lymphocytes and methods of producing and using the same

Abstract

Provided herein are genetically modifying tumor infiltrating lymphocytes (TILs) from patient-derived tumor reactive T cells and methods for generating the same. Also provided herein are compositions of genetically modified TILs and uses of the provided genetically modified TILs for treating cancer in a subject.

Description

GENETICALLY MODIFIED TUMOR INFILITRATING LYMPHOCYTES AND METHODS OF PRODUCING AND USING THE SAMECross-Reference to Related Applications

[0001] This application claims priority from U.S. provisional application No. 63 / 594,407, filed October 30, 2023, entitled “GENETICALLY MODIFIED TUMOR INFILITRATING LYMPHOCYTES AND METHODS OF PRODUCING AND USING THE SAME”, the contents of which are incorporated by reference in their entirety.Incorporation by Reference of Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 220612002040SeqList. xml, created October 28, 2024, which is 176,999 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.Field

[0003] The present disclosure provides genetically modifying tumor infiltrating lymphocytes (TILs) from patient-derived tumor reactive T cells and methods for generating the same. The present disclosure also provides compositions of genetically modified TILs and uses of the provided genetically modified TILs for treating cancer in a subject.Background

[0004] Adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TIL) has emerged as a potential treatment of various types of solid tumors. Clinical studies have demonstrated that T cells isolated from surgically resected tumors possess T-cell receptors (TCRs) that recognize tumor cells and expanding these reactive tumor infiltrating lymphocyte (TIL) populations and re-infusing them into the patient can in some cases result in a dramatic clinical benefit. However, a major obstacle to applications of such cells in cell therapy is maintaining cytotoxicity of the TIL populations. In some cases, responsiveness to TIL therapy can be limited by several mechanisms including tumor mediated- immune suppression, limited persistence, exhaustion, and toxicity associated with high dose IL2 treatment. For example, existing methods for administering TIL therapy often involves administering cytokinesrepeatedly to the patient to maintain cytotoxicity of the TIL populations, however, this can cause toxicity to the subject receiving the administration. Improved methods are needed for maintaining cytotoxicity in TIL populations and cells. Provided herein are embodiments that meet such needs.Summary

[0005] Provided herein is a method of generating genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), the method comprising: (a) selecting cells surface positive for one or more T cell activation markers from a tumor sample from a subject to produce a population of tumor-reactive TIL; (b) activating T cells of the population of tumor-reactive TIL by incubating the T cells with a T cell activation reagent that comprises an anti-CD3 antibody or antigen binding fragment thereof and an anti-CD28 antibody or antigen binding fragment thereof to generate an activated population of tumor-reactive TIL, wherein the activation is carried out for 1 to 6 days; (c) introducing a genetic disruption at a target site within a target gene in T cells of the activated population of tumor-reactive TIL by gene editing to produce a population of genetically modified tumor-reactive TIL; (d) performing an expansion by culturing the population of genetically modified tumor-reactive TIL with one or more T-cell stimulating agents to produce a population of genetically modified and expanded tumor-reactive TIL, wherein one of the one or more T-cell stimulating agents is an anti-CD3 antibody or antigen binding fragment thereof the expansion is carried out for 7 to 21 days; and (e) harvesting the population of genetically modified and expanded tumor-reactive TIL from step (d).

[0006] Provided herein is a method of generating genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), the method comprising: (a) selecting cells surface positive for one or more T cell activation markers from a tumor sample from a subject to produce a population of tumor-reactive TIL; (b) activating T cells of the population of tumor-reactive TIL by incubating the T cells with a T cell activation reagent that comprises an anti-CD3 antibody or antigen binding fragment thereof and an anti-4- IBB antibody or antigen binding fragment thereof to generate an activated population of tumor-reactive TIL, wherein the activation is carried out for 1 to 6 days; (c) introducing a genetic disruption at a target site within a target gene in T cells of the activated population of tumor-reactive TIL by gene editing to produce a population of genetically modified tumor-reactive TIL; (d) performing an expansion byculturing the population of genetically modified tumor-reactive TIL with one or more T-cell stimulating agent to produce a population of genetically modified and expanded tumor-reactive TIL, wherein one of the one or more T-cell stimulating agents is an anti-CD3 antibody or antigen binding fragment thereof the expansion is carried out for 7 to 21 days; and (e) harvesting the population of genetically modified and expanded tumor-reactive TIL from step (d).

[0007] Provided herein is a method of generating genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), the method comprising: (a) obtaining a population of tumor- reactive TIL from a tumor sample from a subject; (b) introducing a genetic disruption at a target site within a target gene selected from the group consisting of SOCS1 , C1SH, PDCD1 (PD-1), TIGIT, RC3HI (Roquin), CMIP (gene encoding C-Maf Inducing Protein), CD39, LAG3. TIM3, MAPK14, and RASA2 (gene encoding RAS p21 protein activator 2)in T cells of the population of tumor-reactive TIL by gene editing to produce a population of genetically modified tumor- reactive TIL; (c) performing an expansion by culturing the population of genetically modified tumor-reactive TIL with one or more T-cell stimulating agent to produce a population of genetically modified and expanded tumor-reactive TIL; and (d) harvesting the population of genetically modified and expanded tumor-reactive TIL from step (c).

[0008] In some embodiments, the population of tumor-reactive TIL are selected TIL in which cells from the tumor sample are selected for cells surface positive for one or more T cell activation marker.

[0009] In some of any embodiments, the one or more T cell activation marker is selected from the group consisting of PD-1, CD39, TIGIT, CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (0X40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM-3 and LAG-3.

[0010] In some of any embodiments, the one or more T cell activation markers comprise CD39 and at least one other marker selected from the group consisting of PD-1, TIGIT, CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (0X40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM-3 and LAG-3.

[0011] In some of any embodiments, the one or more T cell activation markers are PD-1 and / or CD39. In some of any embodiments, the one or more T cell activation markers are PD-1 and CD39.

[0012] In some of any embodiments, the population of tumor-reactive TIL are T cells from the tumor sample further selected for cells surface positive for CD45, CD4 and / or CD8.

[0013] In some embodiments, the population of tumor-reactive TIL is an oligoclonal population that is enriched for T cells expressing a T cell receptor (TCR) reactive to tumor antigen.

[0014] Also provided herein is a method of generating genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), the method comprising: (a) selecting cells surface positive for one or more T cell activation markers from a tumor sample from a subject to produce a population of tumor-reactive TIL; and (b) introducing a genetic disruption at a target site within a target gene selected from the group consisting of SOCS1 , C1SH, PDCD1 (PD-1), TIGIT, RC3HI (Roquin), CMIP (gene encoding C-Maf Inducing Protein), CD39, LAG3. TIM3, MAPK14, and RASA2 (gene encoding RAS p21 protein activator 2) in T cells of the population of tumor-reactive TIL by gene editing to produce a population of genetically modified tumor- reactive TIL; (c) performing an expansion by culturing the population of genetically modified tumor-reactive TIL with one or more T-cell stimulating agent to produce a population of genetically modified and expanded tumor-reactive TIL; and (d) harvesting the population of genetically modified and expanded tumor-reactive TIL from step (c).

[0015] In some embodiments, the one or more T cell activation markers are selected from the group consisting of PD-1, CD39, TIGIT, CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (0X40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM-3 and LAG-3.

[0016] In some of any embodiments, the one or more T cell activation markers comprise CD39 and at least one other marker selected from the group consisting of PD-1, TIGIT, CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (0X40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM-3 and LAG-3.

[0017] In some of any embodiments, the one or more T cell activation markers are PD-1 and / or CD39. In some of any embodiments, the one or more T cell activation markers are PD-1 and CD39.

[0018] In some of any embodiments, in step (a) the cells are further selected for cells positive for the surface marker CD45. In some of any embodiments, in step (a) the cells are further selected for cells positive for the surface marker CD3. In some of any embodiments, instep (a) the cells are further selected for cells positive for the surface markers CD4 and / or CD8. In some of any embodiments, in step (a) the cells are further selected for cells positive for surface markers CD4 and CD8.

[0019] In some of any embodiments, the cells are selected for the marker by contacting cells from the tumor sample with a binding agent that binds to the marker and isolating cells positive for the marker.

[0020] Also provided herein is a method of generating genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), the method comprising: (a) contacting cells from a tumor sample from a subject with a binding agent(s) that detects one or more T cell activation markers; (b) selecting cells from step (a) positive for the one or more T cell activation markers to produce a population of tumor-reactive TIL; (c) introducing a genetic disruption at a target site within a target gene selected from the group consisting of SOCS1 , C1SH, PDCD1 (PD-1), TIGIT, RC3HI (Roquin), CMIP (gene encoding C-Maf Inducing Protein), CD39, LAG3. TIM3, MAPK14, and RASA2 (gene encoding RAS p21 protein activator 2) in T cells of the population of tumor-reactive TIL by gene editing to produce a population of genetically modified tumor- reactive TIL; (d) performing an expansion by culturing the population of genetically modified tumor-reactive TIL with one or more T-cell stimulating agent to produce a population of genetically modified and expanded tumor-reactive TIL; and (e) harvesting the population of genetically modified and expanded tumor-reactive TIL from step (d).

[0021] In some embodiments, the one or more T cell activation markers are selected from the group consisting of PD-1, CD39, TIGIT, CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (0X40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM-3 and LAG-3.

[0022] In some of any embodiments, the one or more T cell activation markers comprise CD39 and at least one other marker selected from the group consisting of PD-1, TIGIT, CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (0X40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM-3 and LAG-3.

[0023] In some of any embodiments, the one or more T cell activation markers are PD-1 and / or CD39. In some of any embodiments, the one or more T cell activation markers are PD-1 and CD39.

[0024] In some of any embodiments, in step (a) the cells from the tumor sample are further contacted with a binding agent that detects CD45 and in step (b) selecting cells surface positive for CD45.

[0025] In some of any embodiments, in step (a) the cells are further contacting with a binding agent that detects CD3 and in step (b) selecting for cells surface positive for CD3.

[0026] In some of any embodiments, in step (a) the cells are further contacted with binding agent(s) that detect CD4 and / or CD8 and in step (b) selecting for cells surface positive for CD4 and / or CD8. In some of any embodiments, in step (a) the cells are further contacted with binding agent(s) that detects CD4 and CD8 and in step (b) selecting for cells surface positive for CD4 and CD 8.

[0027] In some of any embodiments, prior to introducing the genetic disruption, the method comprises incubating the population of tumor-reactive TIL with a T cell activation reagent to produce a population of activated TIL.

[0028] In some embodiments, the T cell activation reagent comprises an anti-CD3 antibody or antigen binding fragment thereof.

[0029] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof is a primary T cell activation agonist, and the T cell activation reagent further comprises a costimulatory agonist.

[0030] In some embodiments, the costimulatory agonist is an anti-CD28 antibody or antigen binding fragment thereof.

[0031] In some embodiments, the costimulatory agonist is an anti-4- IBB antibody or antigen binding fragment thereof.

[0032] In some embodiments, the T cell activation reagent comprises an anti-CD3 antibody or antigen binding fragment thereof and an anti-CD28 antibody or antigen binding fragment thereof.

[0033] In some of any embodiments, the T cell activation reagent comprises an anti-CD3 antibody or antigen binding fragment thereof and an anti-4- IBB antibody or antigen binding fragment thereof.

[0034] In some of any embodiments, the incubating of the population of tumor-reactive TIL with the T cell activation reagent takes place for about 1 days to 5 days.

[0035] In some of any embodiments, the incubating of the population of tumor-reactive TIL with the T cell activation reagent takes place for about 2 days to 4 days.

[0036] In some of any embodiments, the incubating of the population of tumor-reactive TIL with the T cell activation reagent takes place for about 3 days.

[0037] In some of any embodiments, the incubating of the population of tumor-reactive TIL with the T cell activation reagent is performed without feeder cells.

[0038] In some of any embodiments, prior to introducing the genetic disruption, the method comprises incubating the population of tumor-reactive TIL with a first T cell activation reagent to produce a first activated population of TILs and incubating the first activated population of TIL with a second T cell activation reagent for producing a second activated population of TIL.

[0039] In some embodiments the first T cell activation reagent comprises an anti-CD3 antibody or antigen binding fragment thereof and an anti-4- IBB antibody or antigen binding fragment thereof and / or a 4- IBB agonist.

[0040] In some of any embodiments, the second T cell activation reagent comprises an anti- CD3 antibody or antigen binding fragment thereof.

[0041] In some embodiments, the second T cell activation reagent further comprises an anti- CD28 antibody.

[0042] In some embodiments, the second T cell activation reagent further comprises an anti- 4-1BB antibody and / or 4-1BB agonist.

[0043] Provided herein is a method of generating genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), the method comprising: (a) selecting cells surface positive for one or more T cell activation markers from a tumor sample from a subject to produce a population of tumor-reactive TIL; (b) incubating T cells of the population of tumor-reactive TIL by incubating the T cells with a first T cell activation reagent that comprises an anti-CD3 antibody or antigen binding fragment thereof and an anti-4- IBB antibody or antigen binding fragment thereof to generate a first activated population of TIL; (c) incubating the T cells of the first activated population of TIL with a second T cell activation reagent that comprises an anti- CD3 antibody or antigen binding fragment thereof and an anti-CD28 antibody or antigen binding fragment thereof to generate a second activated population of TIL; (d) introducing a genetic disruption at a target site within a target gene in T cells of the second activated population of TIL by gene editing to produce a population of genetically modified tumor- reactive TIL; (e) performing an expansion by culturing the population of genetically modified tumor-reactive TIL with one or more T-cell stimulating agent to produce a population of genetically modified and expanded tumor-reactive TIL, wherein one of the one or more T-cellstimulating agents is an anti-CD3 antibody or antigen binding fragment thereof the expansion is carried out for 7 to 21 days; and (f) harvesting the population of genetically modified and expanded tumor-reactive TIL from step (d).

[0044] In some of any embodiments, the incubating of the population of tumor-reactive TIL with the first T cell activation reagent takes place for about 2 days to 14 days.

[0045] In some of any embodiments, the incubating of the population of tumor-reactive TIL with the first T cell activation reagent takes place for about 10 days to 12 days.

[0046] In some of any embodiments, the incubating of the population of tumor-reactive TIL with the first T cell activation reagent takes place for about 11 days.

[0047] In some of any embodiments, the incubating of the population of tumor-reactive TIL with the first T cell activation reagent takes place for about 6 days to 8 days.

[0048] In some of any embodiments, the incubating of the population of tumor-reactive TIL with the first T cell activation reagent takes place for about 7 days.

[0049] In some of any embodiments, incubating of the first activated population of TIL with the second T cell activation reagent takes place for about 2 days to 4 days.

[0050] In some of any embodiments, incubating the first activated population of TIL with the second T cell activation reagent takes place for about 3 days.

[0051] In some of any embodiments, incubating the population of tumor-reactive TIL with the first T cell activation reagent and / or incubating the first activated population of TIL with the second T cell activation reagent is performed without feeder cells.

[0052] In some of any embodiments, incubating the population of tumor-reactive TIL with the T cell activation reagent is carried out in the presence of recombinant IL-2.

[0053] In some of any embodiments, the incubating the population of tumor-reactive TIL with the first T cell activation reagent and / or the incubating the first activated population of TIL with the second T cell activation reagent is carried out in the presence of recombinant IL-2.

[0054] In some of any embodiments, the recombinant IL-2 is at a concentration of between at or about 1000 lU / mL to 6,000 lU / mL, optionally at a concentration of about 3,000 lU / mL.

[0055] In some of any embodiments, the activated population of tumor-reactive TIL is introduced with the genetic disruption within about 3 days of incubating with the first T cell activation reagent.

[0056] In some of any embodiments, the second activated population of TIL is introduced with the genetic disruption within about 3 days of incubating with the second T cell activation reagent.

[0057] In some of any embodiments, the genetic disruption is introduced using a gene editing agent comprising a CRISPR-Cas combination comprising a guide RNA (gRNA) that binds to the target site and a Cas nuclease.

[0058] In some embodiments, the Cas nuclease is a Cas9. In some embodiments, the Cas9 is a streptococcus pyogenes Cas9 (spCas9). In some embodiments, the Cas nuclease is Mad7.

[0059] In some of any embodiments, the gene editing agent is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease.

[0060] In some of any embodiments, the gene editing agent is a delivery vector comprising a polynucleotide encoding the Cas9 nuclease and comprising the gRNA.

[0061] In some embodiments, the delivery vector is a lipid nanoparticle or is a viral vector.

[0062] In some of any embodiments, the gene editing agent is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease.

[0063] In some embodiments, the RNP complex is delivered to the T cells of the population of tumor-reactive TIL using electroporation.

[0064] In some embodiments, the RNP complex is delivered to the T cells of the population of tumor-reactive TIL using an amphiphilic peptide reagent.

[0065] In some embodiments, the RNP complex is mixed with the amphiphilic peptide reagent prior to the introducing step.

[0066] In some of any embodiments, the RNP complex is delivered using peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC).

[0067] In some of any embodiments, the target gene is SOCS1 , C1SH or PD-1.

[0068] In some of any embodiments, the genetic disruption is at a target site within the SOCS1 locus. In some embodiments, the genetic disruption is at a target site within exon 2. In some of any embodiments, the genetic disruption is in the SH2 domain. In some of any embodiments, the genetic disruption is in a target site within genomic coordinates chl6:ll, 255, 013-11, 255, 255.

[0069] In some of any embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOS: 13-17, optionally wherein the target site is set forth in any one of SEQ ID Nos: 13-17. In some of any embodiments, the target site comprises the sequence set forth inSEQ ID NO: 13, optionally wherein the target site is set forth in SEQ ID NO: 13. In some of any embodiments, the target site comprises the sequence set forth in SEQ ID NO: 14, optionally wherein the target site is set forth in SEQ ID NO: 14.

[0070] In some of any embodiments, the gRNA comprises a spacer sequence set forth in any one of SEQ ID NOS: 35-39. In some of any embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 35. In some of any embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 36.

[0071] In some of any embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOS: 21-22, optionally wherein the target site is set forth in SEQ ID NO: 21 or SEQ ID NO:22.

[0072] In some of any embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 43 or SEQ ID NO: 44.

[0073] In some of any embodiments, the genetic disruption is in the KIR domain. In some of any embodiments, the genetic disruption is in a target site within genomic coordinates chl6:l l, 255, 287-11, 255, 329.

[0074] In some of any embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOS: 18-20, optionally wherein the target site is set forth in any one of SEQ ID Nos: 18-20. In some of any embodiments, the target site comprises the sequence set forth in SEQ ID NO: 20, optionally wherein the target site is set forth in SEQ ID NO: 20.

[0075] In some of any embodiments, the gRNA comprises a spacer sequence set forth in any one of SEQ ID NOS: 40-42. In some of any embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 42.

[0076] In some of any embodiments, the genetic disruption is at a target site within the C1SH locus. In some embodiments, the genetic disruption is at a target site within exon 3. In some of any embodiments, the genetic disruption is in a target site within genomic coordinates ch3: 50,607,687-50,608,132.

[0077] In some of any embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOS: 1-7, optionally wherein the target site is set forth in any one of SEQ ID NOS: 1-7. In some of any embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOS: 1-5, optionally wherein the target site is set forth in any one of SEQ ID NOS: 1-5. In some of any embodiments, the target site comprises the sequence set forth in SEQ ID NO: 5, optionally wherein the target site is set forth in SEQ ID NO:5.

[0078] In some of any embodiments, the gRNA comprises a spacer sequence set forth in any one of SEQ ID NOS: 23-29.

[0079] In some of any embodiments, the gRNA comprises a spacer sequence set forth in any one of SEQ ID NOS: 23-27. In some of any embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 27.

[0080] In some of any embodiments, the genetic disruption is at a target site within the PD-1 locus. In some embodiments, the genetic disruption is at a target site within exon 2, 3 or 5. In some of any embodiments, the genetic disruption is in a target site within genomic coordinates ch2:241, 852, 672-241, 851, 173.

[0081] In some of any embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOS: 8-12, optionally wherein the target site is set forth in any one of SEQ ID NOS: 8-12. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 10, optionally wherein the target site is set forth in SEQ ID NO: 10.

[0082] In some of any embodiments, the gRNA comprises a spacer sequence set forth in any one of SEQ ID NOS: 30-34. In some of any embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO:32.

[0083] In some of any embodiments, the genetic disruption comprises an indel in the target gene in cells of population of genetically modified cells.

[0084] In some of any embodiments, the genetic disruption inactivates all alleles of the target gene in cells of population of genetically modified cells.

[0085] In some of any embodiments, the genetic disruption is a knockout of the target gene in cells of population of genetically modified cells.

[0086] In some of any embodiments, the one or more T-cell stimulating agent of lymphocytes is an anti-CD3 agent (e.g. OKT3) and / or a recombinant cytokine selected from one or more of IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27 and IL-35.

[0087] In some of any embodiments, the one or more T-cell stimulating agents of lymphocytes comprises one or more recombinant cytokines selected form IL-2, IL15, and IL-21.

[0088] In some of any embodiments, at least one of the one or more T-cell stimulating agent is recombinant IL-2.

[0089] In some of any embodiments, the concentration of recombinant IL-2 added to the culture for the expansion is from 100 lU / mL to 6000 lU / mL, such as from 300 lU / mL to 3000lU / mL. In some embodiments, the concentration of IL-2 added to the culture for the expansion is at or about 3000 lU / mL.

[0090] In some of any embodiments, the one or more T-cell stimulating agents does not include recombinant IL-2. In some of any embodiments, the expansion is performed without the presence of recombinant IL-2.

[0091] In some of any embodiments, at least one of the T-cell stimulating agent is IL-15 and / or IL-21.

[0092] In some of any embodiments, the expansion is performed in the presence of IL- 15 and IL-21.

[0093] In some of any embodiments, the concentration of IL- 15 added to the culture for expansion is from 250 lU / mL to 1500 lU / mL, such as from 250 lU / mL to 750 lU / mL.

[0094] In some embodiments, the concentration of IL- 15 added to the culture for the expansion is at or about 500 lU / mL.

[0095] In some of any embodiments, the concentration of IL-21 added to the culture for expansion is from 0.5 lU / mL to 10 lU / mL, such as from 1 lU / mL to 5 lU / mL.

[0096] In some of any embodiments, the concentration of IL-21 added to the culture for the expansion is at or about 1.15 lU / mL.

[0097] In some of any embodiments, at least one of the T-cell stimulating agent is an anti- CD3 antibody, optionally wherein the anti-CD3 antibody is OKT3.

[0098] In some of any embodiments, the anti-CD3 antibody is added to the culture at a concentration of from about 10 ng / mL to about 100 ng / mL, such as at a concentration of from about 20 ng / mL to about 50 ng / mL.

[0099] In some of any embodiments, the anti-CD3 antibody is added to the culture at a concentration of about 30 ng / mL.

[0100] In some of any embodiments, the one or more T-cell stimulating agent further comprises feeder cells that are non-dividing peripheral blood mononuclear cells (PBMC), optionally wherein the feeder cells are gamma irradiated.

[0101] In some embodiments, the feeder cells are present at a ratio of feeder cells to TIL cells of from about 50:1 to 500:1, optionally wherein the ratio of feeder cells to TIL cells is at or about 200:1.

[0102] In some of any embodiments, the one or more T cell stimulating agent comprises about 3000 lU / mL IL-2, 30 ng / mL anti-CD3 antibody (OKT3) and gamma irradiated PBMC feeder cells at a ratio of about 200: 1 feeder cells to TIL.

[0103] In some of any embodiments, the performing the expansion to produce the expanded population of T cells is for 7 to 35 days.

[0104] In some of any embodiments, the performing the expansion to produce the expanded population of T cells is for 7 to 28 days, optionally 14 days to 28 days.

[0105] In some of any embodiments, the performing the expansion to produce the expanded population of T cells is for 7 to 21 days, optionally 7 to 14 days.

[0106] In some of any embodiments, the expansion is a second expansion, and the method comprises performing a first expansion prior to introducing the genetic disruption at a target site in T cells of the population of tumor-reactive TIL, wherein the first expansion is by culturing the population of tumor-reactive TIL with one or more first T-cell stimulating agent.

[0107] In some of any embodiments, the expansion is a second expansion, and the method comprises performing a first expansion prior to the second expansion but subsequent to the introducing a genetic disruption, wherein the first expansion is by culture of the population of tumor-reactive TIL with one or more first T-cell stimulating agent.

[0108] In some embodiments, the one or more first T-cell stimulating agent of lymphocytes is an anti-CD3 agent (e.g. OKT3) and / or a recombinant cytokine selected from one or more of IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27 and IL-35.

[0109] In some of any embodiments, at least one of the one or more first T-cell stimulating agent is recombinant IL-2.

[0110] In some of any embodiments, the concentration of recombinant IL-2 added to the culture for the first expansion is from 100 lU / mL to 6000 lU / mL, such as from 300 lU / mL to 3000 lU / mL.

[0111] In some embodiments, the concentration of IL-2 added to the culture for the first expansion is at or about 3000 lU / mL.

[0112] In some of any embodiments, at least one of the T-cell stimulating agent is IL-15 and / or IL-21.

[0113] In some embodiments, the concentration of IL- 15 added to the culture for the first expansion is from 250 lU / mL to 1500 lU / mL, such as from 250 lU / mL to 750 lU / mL.

[0114] In some of any embodiments, the concentration of IL- 15 added to the culture for the first expansion is at or about 500 lU / mL.

[0115] In some of any embodiments, the concentration of IL-21 added to the culture for the first expansion is from 0.5 lU / mL to 10 lU / mL, such as from 1 lU / mL to 5 lU / mL.

[0116] In some of any embodiments, the concentration of IL-21 added to the culture for the first expansion is at or about 1.15 lU / mL.

[0117] In some of any embodiments, at least one of the first T-cell stimulating agent is an anti-CD3 antibody, optionally wherein the anti-CD3 antibody is OKT3.

[0118] In some embodiments, the anti-CD3 antibody is added to the culture at a concentration of from about 10 ng / mL to about 100 ng / mL, such as at a concentration of from about 20 ng / mL to about 50 ng / mL.

[0119] In some embodiments, the anti-CD3 antibody is added to the culture at a concentration of about 30 ng / mL.

[0120] In some of any embodiments, the one or more first T-cell stimulating agent further comprises feeder cells that are non-dividing peripheral blood mononuclear cells (PBMC), optionally wherein the feeder cells are gamma irradiated.

[0121] In some of any embodiments, the feeder cells are present at a ratio of feeder cells to TIL cells of from about 50:1 to 500:1, optionally wherein the ratio of feeder cells to TIL cells is at or about 200: 1.

[0122] In some of any embodiments, the one or more first T cell stimulating agents comprise about 3000 lU / mL IL-2, 30 ng / mL anti-CD3 antibody (OKT3) and gamma irradiated PBMC feeder cells at a ratio of about 200: 1 feeder cells to TIL.

[0123] In some of any embodiments, the performing the first expansion to produce the expanded population of T cells is for 7 to 35 days.

[0124] In some of any embodiments, the performing the expansion to produce the expanded population of T cells is for 7 to 28 days, optionally 14 days to 28 days.

[0125] In some of any embodiments, the performing the expansion to produce the expanded population of T cells is for 7 to 21 days, optionally 7 to 14 days.

[0126] In some of any embodiments, the input sample comprising T cells is derived from a resected tumor.

[0127] In some embodiments, the input sample comprising T cells is a single cell suspension processed by homogenization and / or enzymatic digestion of one or more tumor fragments from the resected tumor.

[0128] In some of any embodiments, the input sample comprising T cells is a single cell suspension processed by homogenization and enzymatic digestion of one or more tumor fragments from the resected tumor.

[0129] In some of any embodiments, the enzymatic digestion is by incubation with a collagenase, optionally collagenase IV or collagenase I / II.

[0130] In some of any embodiments, the selecting is carried out simultaneously or sequentially in any order to obtain the population of tumor-reactive TIL.

[0131] In some of any embodiments, the selecting is by flow cytometry.

[0132] In some embodiments, selecting the cells comprises sorting the cells positive for the at least 4 fluorescence signal based on a fluorescence minus one (FMO) cocktail.

[0133] In some of any embodiments, the sorting is performed at a rate of 5,000 events per second to 10,000 events per second, optionally at about 6,000 events per second.

[0134] In some of any embodiments, selecting cells is performed using a microfluidics chip based cell sorting comprising at least 4 fluorescence detectors.

[0135] In some of any embodiments, the performing the expansion is carried out in a closed system using a gas permeable membrane. In some of any embodiments, the performing the expansion is carried out in a closed system using a bioreactor.

[0136] In some of any embodiments, the performing the first expansion is carried out in a closed system using a gas permeable culture vessel. In some of any embodiments, the performing the first expansion is carried out in a closed system using a bioreactor.

[0137] In some of any embodiments, the performing the second expansion is performed in a gas permeable culture vessel. In some of any embodiments, the performing the second expansion is performed using a bioreactor.

[0138] In some of any embodiments, the tumor is a tumor of an epithelial cancer.

[0139] In some of any embodiments, the tumor is a tumor of a melanoma, lung squamous, lung adenocarcinoma, bladder cancer, lung small cell cancer, esophageal cancer, colorectal cancer (CRC), cervical cancer, head and neck cancer, stomach cancer or uterine cancer.

[0140] In some of any embodiments, the tumor is a melanoma.

[0141] In some of any embodiments, the tumor is a colorectal cancer (CRC).

[0142] In some of any embodiments, the tumor is a tumor of a non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma cancer, endometrial cancer, optionally wherein the breast cancer is HR+ / Her2- breast cancer, triple negative breast cancer (TNBC) or HER2+ breast cancer.

[0143] Provided herein is a composition of genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs) produced by the method of some of any embodiments.

[0144] Provided herein is a SOCS-1 targeting gRNA comprising a spacer sequence set forth in any one of SEQ ID NOS: 35-44.

[0145] In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 35. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 36. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 42. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 43. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 44.Provided herein is a CISH-targeting gRNA comprising a spacer sequence set forth in any one of SEQ ID NOS: 23-29.

[0146] In some embodiments, the gRNA comprises a spacer sequence set forth in any one of SEQ ID NOS: 23-27.

[0147] In some of any embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 27.

[0148] Provided herein is a PD-1 -targeting gRNA comprising a spacer sequence set forth in any one of SEQ ID NOS: 30-34.

[0149] In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO:32.

[0150] In some of any embodiments, the gRNA further comprises a scaffold sequence (tracR RNA) for complexing with a Cas protein.

[0151] Provided herein is a CRISPR-Cas combination comprising a guide RNA (gRNA) of some of any embodiments and a Cas nuclease.

[0152] In some embodiments, the Cas nuclease is a Cas9.

[0153] In some embodiments, the Cas9 is a streptococcus pyogenes Cas9 (spCas9).

[0154] In some embodiments, the Cas nuclease is Mad7.

[0155] In some of any embodiments, the gene editing agent is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease.

[0156] Provided herein is a vector comprising the CRISPR-Cas combination of some of any embodiments.

[0157] In some embodiments, the vector is a lipid nanoparticle or is a viral vector.

[0158] Provided herein is a composition comprising a population of genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), wherein at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the total cells or total T cells in the composition comprise a genetic disruption at a target site in a SOCS1 target gene.

[0159] Provided herein is a composition comprising a population of genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), wherein at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the total cells or total T cells in the composition comprise a genetic disruption at a target site in a C1SH target gene.

[0160] Provided herein is a composition comprising a population of genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), wherein at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the total cells or total T cells in the composition comprise a genetic disruption in a target site in a PDCD1 (PD-l)target gene.

[0161] In some of any embodiments, the genetic disruption is an indel.

[0162] In some of any embodiments, the genetic disruption inactivates all alleles of the target gene.

[0163] In some of any embodiments, the genetic disruption knock out expression of the target gene, optionally wherein expression is genomic expression or is protein expression on the cell surface as determined by flow cytometry.

[0164] In some of any embodiments, at least 90% of cells in the composition are CD3+ T cells and less than about 5% of the population are T regulatory cells.

[0165] In some of any embodiments, the population of genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs) are an oligoclonal population.

[0166] In some embodiments, up to 40 clones make up at least 40% of the TCR frequency in the population.

[0167] In some of any embodiments, the pharmaceutical composition is for treatment of a patient’s tumor.

[0168] In some embodiments, the tumor is a kidney, lung, colorectal cancer (CRC) tumor, a melanoma tumor, a non-small cell lung cancer (NSCLC) tumor, or an ovarian cancer tumor.

[0169] In some of any embodiments, the tumor is from a human subject.

[0170] In some embodiments, the pharmaceutical composition is for autologous adoptive therapy to the human subject.

[0171] In some of any embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

[0172] In some of any embodiments, further comprising a cryoprotectant.

[0173] In some of any embodiments, the composition is a liquid composition.

[0174] In some embodiments, the composition had been frozen and thawed.

[0175] In some of any embodiments, the volume of the composition is between 1 mL and 500 mL.

[0176] In some of any embodiments, the composition is frozen.

[0177] Provided herein is a method of treating a solid tumor malignancy in a subject, the method comprising administering a T lymphocyte infiltrating (TIL) composition of some of any embodiment to the subject.

[0178] In some embodiments, the tumor is a tumor of an epithelial cancer.

[0179] In some of any embodiments, the tumor is a tumor of a lung (e.g., lung squamous, lung adenocarcinoma, lung small cell cancer), kidney, melanoma, bladder cancer, esophageal cancer, colorectal cancer (CRC), cervical cancer, head and neck cancer, stomach cancer or uterine cancer.

[0180] In some of any embodiments, the tumor is a tumor of a non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma cancer, endometrial cancer, optionally wherein the breast cancer is HR+ / Her2- breast cancer, triple negative breast cancer (TNBC) or HER2+ breast cancer.

[0181] In some of any embodiments, the solid tumor malignancy is selected from the group comprising: breast cancer, colorectal carcinoma (CRC), uveal melanoma, cutaneous carcinoma, non-small cell lung cancer, head and neck squamous cell carcinoma, or any advanced solid tumor.

[0182] In some of any embodiments, the method further comprises administration of a lymphodepleting therapy.

[0183] In some embodiments, the lymphodepleting therapy is or comprises cyclophosphamide, ifosfamide, altretamine, busulfan, carboplatin, carmustine, cisplatin, dacarbazine, lomustine, melphalan, temozolomide, trabectedin, sodium 2-mercaptoethane sulfonate (Mensa), fludarabine, floxuridine, or any combination thereof.

[0184] In some of any embodiments, the lymphodepleting therapy is or comprises cyclophosphamide, sodium 2-mercaptoethane sulfonate (Mensa), fludarabine, or a combination thereof.

[0185] In some of any embodiments, the lymphodepleting therapy comprises administration of cyclophosphamide from about 30 mg / kg to about 200 mg / kg, optionally wherein the cyclophosphamide is administered at or about 60 mg / kg, optionally wherein the cyclophosphamide is administered within about 5 to 9 days prior to administering the TIL composition, more optionally wherein the cyclophosphamide is administered about 7 days prior to administering the TIL composition (Day -7).

[0186] In some of any embodiments, the lymphodepleting therapy comprises administration of Mensa from about 5 mg / kg / day to 25 mg / kg / day, optionally wherein the Mensa is administered at or about 15 mg / kg / day, optionally wherein the Mensa is administered within about 4 to 8 days prior to administering the TIL composition, more optionally wherein the Mensa is administered about 6 days prior to administering the TIL composition (Day -6).

[0187] In some of any embodiments, the lymphodepleting therapy comprises administration of fludarabine from about 20-40 mg / m2, optionally at or about 25 mg / m2daily, optionally wherein the fludarabine is administered for three consecutive days, optionally wherein the fludarabine is administered 3 to 5 days prior to administering the TIL composition (Day -5 to Day -3).

[0188] In some of any embodiments, the administration of the lymphodepleting therapy is completed within about 2 to 7 days prior to initiation of the administration of the TIL composition, optionally 2 to 5 days prior to initiation of the administration of the TIL composition.

[0189] In some of any embodiments, further comprising administering recombinant IL-2 to the subject.

[0190] In some embodiments, the initiation of administration of the recombinant IL-2 is 2 hour to 24 hours after administration of the TIL composition, optionally wherein initiation of administration of the recombinant IL-2 is 2-3 hours after administration of the TIL composition.

[0191] In some of any embodiments, each dose of the recombinant IL-2 is from 600,000 lU / kg and 1,000,000 lU / kg, optionally administered 3 to 4 times a day for up to 3 days.

[0192] In some of any embodiments, the recombinant IL-2 is Aldesleukin.

[0193] In some of any embodiments, the TIL composition is administered without administering recombinant IL-2 to the subject.

[0194] In some embodiments, the TIL composition comprises a population of genetically modified TIL with a genetic disruption of the SOCS1 locus.

[0195] In some of any embodiments, the target gene is selected from the group consisting of SOCS1, CISH, PDCD1 (PD-1), TIGIT, RC3HI (Roquin), CMIP (gene encoding C-Maf Inducing Protein), CD39, LAG3, TIM3, MAPK14, and RASA2 (gene encoding RAS p21 protein activator 2).Brief Description of the Drawings

[0196] FIG. 1 depicts a western blot of CISH protein levels in primary human T cells for unedited T cells (mock) or CISH KO T cells.

[0197] FIG. 2A-2F depicts the gene editing efficiency of TIL transfected with SOCS1, CISH or a non-targeting control sgRNA and rapidly expanded for 14 days with 1:200 irradiated- PBMCs, 30ng / ml of OKT3 and either 3000 lU / ml of IL2 or 50ng / ml of IL15 and IL21 (500 lU / mL of IL- 15 and 1.15 lU / mL of IL-21). FIG. 2A depicts the frequency of TILs after 14 days of REP for unedited TIL or PD-1 KO TIL that stain positive for PD-1 using flow cytometry. FIG. 2B depicts the frequency of TILs after 14 days of REP for unedited TIL or LAG3 KO TIL that stain positive for LAG3 using flow cytometry. FIG. 2C depicts the frequency of TILs after 14 days of REP for unedited TIL or TIGIT KO TIL that stain positive for TIGIT using flow cytometry. FIG. 2D depicts the frequency of TILs after 14 days of REP for unedited TIL or TIM3 KO TIL that stain positive for TIM3 using flow cytometry. FIG. 2E depicts the indel and knockout frequency of TILs after 14 days of REP for unedited TIL or edited TIL (SOCS1 KO TIL or CISH KO TIL), with the SOCS1 KO TIL expanded in IL- 15 and IL-21 instead of IL-2 for the second rapid expansion FIG. 2F depicts a western blot of SOCS1 in TILs after 14 days of REP for unedited TIL and SOCS1 KO TILs.

[0198] FIG. 3A-3C depicts cell yield and phenotype in TILs after 14 days of REP for unedited TIL and edited TIL (PD-1 KO TIL, CISH KO TIL, or SOCS1 TIL). FIG. 3A depicts the cell yield during the 14 day REP for unedited TIL and edited TIL (PD-1 KO TIL and SOCS1 TIL). FIG. 3B depicts the percent of live cells at the end of the 14 REP for unedited TIL and edited TIL (PD-1 KO TIL, CISH KO TIL, and SOCS1 TIL). FIG. 3C depicts thepercentage (%) of CD3+ cells that are different phenotypes after the 14 day REP for unedited TIL and edited TIL (PD-1 KO TIL and SOCS1 TIL).

[0199] FIG. 4 depicts the polyfunctionality of TILs after 14 days of REP for unedited TIL and edited TIL (PD-1 KO TIL, CISH KO TIL, and SOCS1 TIL) by staining the TILs for IFNy, TNEa, IL-2, and CD107a and determining the percentage of cells positive for 0, 1, 2, 3, or all 4 markers.

[0200] FIG. 5A-5D depicts the cytolysis, expansion, and IFNy secretion of TILs after 14 days of REP for unedited TIL and edited TIL (SOCS1 KO TIL). FIG. 5A depicts images of unedited TIL or edited TIL (SOCS1 KO TIL) co-cultured with tumor cells. FIG. 5B depicts the percent (%) cytolysis of unedited TIL and edited TIL (SOCS1 KO TIL) co-cultured with tumor cells and normalized to tumor cells cultured alone without TIL present. FIG. 5C depicts the total number of CD3+ cells after 14 days of REP for unedited TIL and edited TIL (SOCS1 KO TIL). FIG. 5D depicts the concentration of IFNy present in the supernatant of cultures with either tumor alone or tumor co-cultured with either unedited TIL or edited TIL (SOCS1 KO TIL).

[0201] FIGS. 6A-6C depict results from PDl+CD39+-derived TIL transfected with sgRNA targeting SOCS1 or a non-targeting control sgRNA and rapidly expanded for 14 days with 1:200 irradiated-PBMCs, 30ng / ml of OKT2 and either 3000 lU / ml of IL2 or 50ng / ml of IL15 and IL21. FIG. 6A depicts SOCS knockout (KO) frequency of TIL at 72 hours post-transfection and after 14 days of rapid expansion (REP) in IL2 or IL15 / IL21. FIG. 6B depicts cell counts of TILs after 14 days of REP in IL2 or IL15 / 1L21 for unedited TIL (PD1+CD39+ TIL) or edited TIL (SOCS1 KO PD1+CD39+ TIL). FIG. 6C depicts CD8 frequency of unedited TIL (PD1+CD39+ TIL) or edited TIL (SOCS1 KO PD1+CD39+ TIL) after 14 days of rapid expansion (REP) in IL2 or IL15 / IL21.

[0202] FIG. 7A depicts the gene editing efficiency of MART- 1 specific TILs after 14 days of REP for unedited TIL (PD1+CD39+ TIL) and edited TIL (PD-1 KO PD1+CD39+ TIL, CISH KO PD1+CD39+ TIL, and SOCS1 PD1+CD39+ TIL) measured by both indel and knockout frequency.

[0203] FIG. 7B is a schematic of the MARTI serial killing assay for assessing TIL cytolysis and expansion. MeWo (melanoma) target cells expressing the MARTI reactive antigen (green) are co-cultured with TIL (blue) for 3 days, and subsequently transferred to a new culture containing pre-seeded MeWo cells. This was repeated 3 times. Tumor cell confluency wasmonitored throughout the assay by imaging and impedance biosensor measurements using the eSIGHT.

[0204] FIG. 7C depicts cytolysis data from Round 1 and Round 4 of a serial killing assay as described in FIG. 2A, wherein TIL is co-cultured with MeWO cells with either no cytokine support or with 30IU / ml of IL2. Cytolysis was calculated based on impedance using the eSITE.

[0205] FIG. 7D depicts cytolysis of MeWo target cells during consecutive co-culture with SOCS1KO (green), CISH KO (grey), PD-1 KO (blue) or non-targeted control (black) TIL at a 3:1 effector to target ratio in the absence of IL2 (n=3).

[0206] FIG. 7E depicts representative images of MeWo target cells labeled with nuclight red at 0, 10, and 19.5 hours of co-culture with control, SOCS1 KO, and CISH KO MARTI reactive TIL during round 2 of stimulation.

[0207] FIG. 7F depicts impedance-based cytolysis of MeWo target cells by non-targeting control, SOCS1 KO, and CISH KO MARTI reactive TIL at 3:1 effector to target ratio through 4 consecutive rounds of the serial killing assay in the presence of IL2.

[0208] FIG. 7G depicts fold change in TIL expansion relative to day 0. CD8+ T cells were quantified at the start and end the serial killing assay depicted in EIG. 2A using flow cytometry.

[0209] FIG. 8A and FIG. 8B depict the average sum intensity of Cleaved-Caspase 3 expression in patient-derived melanoma cells after co-culture with autologous SOCS1 KO, CISH KO or control TIL (nontargeting sgRNA) for 24, 48, and 72 hours in the presence or absence of HLA-I and HLA-II block without IL2 support (EIG. 3A) or with 300 lU / mL of IL-2 added to the co-culture (EIG. 3B). The average sum intensity was normalized against the average sum intensity of Cleaved-Caspase 3 / 7 expression in the tumor only control. Tumor cell killing was monitored at 24 hours intervals by imaging with the cytation 5.

[0210] FIG. 9A-9E depict a schematic of a gene editing method with an initial anti- CD3 / anti-4-lBB stimulation and one round of REP (as depicted in FIG. 9A) and the results. The results for editing efficiency (by percent indel and percent knockout expression) for both PD1+CD39+ double positive cells and SP / DN cells isolated from both kidney tumor samples at the end of REP 2 is shown in FIG. 9B. FIG. 9C shows the cell number measured using a cellometer at the end of REP2 for TIL that had CISH knocked out or were treated with a nontargeting control sgRNA (NTC) in PD1+CD39+ or SP / DN cells isolated from both kidney tumors. The proportion of TIL that were CD4+ and / or CD8+ was measured at the end of the second REP in both SP / DN cells and PD1+CD39+ cells that were edited with a non-targetingcontrol sgRNA (NTC) or were edited to knockout CISH in cells isolated from the first kidney tumor (shown in FIG. 9D) and the second kidney tumor (shown in FIG. 9E).

[0211] FIG. 10A-10F shows an exemplary method of genetic engineering. FIG. 10A depicts the exemplary method of genetic engineering of TIL. Editing efficiency by percent (%) indel for both PD1+CD39+ double positive cells and SP / DN cells isolated from all three kidney tumor samples at the end of the REP is shown in FIG. 10B, while the percent (%) knockout is shown in FIG. 10C using the method depicted in FIG. 10A. The fold change in cell numbers after the REP compared to the cell numbers after the initial anti-CD3 / anti-4-lBB activation are shown in FIG. 10D. FIG. 10E shows the proportion of TIL that were CD4+ and / or CD8+ at the end of the REP in both SP / DN cells and PD1+CD39+ cells that were edited with a non-targeting control sgRNA (NTC) or were edited to knockout CISH or SOCS1 in cells isolated from the two of the kidney tumors. FIG. 10F shows the proportion of TIL that were stained with CCR7 and / or CD45RA at the end of the REP in both SP / DN cells and PD1+CD39+ cells that were edited with a non-targeting control sgRNA (NTC) or were edited to knockout CISH or SOCS1 in cells isolated from the two of the kidney tumors to obtain memory phenotype of the cells.

[0212] FIG. 11A-11C depict an exemplary method of genetic engineering TIL. FIG. 11A depict a schematic of the exemplary method. FIG. 1 IB shows the results for editing efficiency by percent (%) indel and percent (%) knockout for both PD1+CD39+ double positive cells and SP / DN cells isolated from the kidney tumor and the lung tumor samples at the end of the REP. FIG. 11C shows the cell number at the end of the REP after gene editing for TIL that had SOCS1 knocked out using either the sgRNA SOCS1#1 (labeled as SOCS1) or the sgRNA sgKIR(SOCSl)#3 (labeled as SOCS1-KIR), or were treated with a non-targeting control sgRNA (labeled as sgNTC) in PD1+CD39+ or SP / DN cells isolated from either the kidney or the lung tumors.

[0213] FIG. 12A-12E depict results of exemplary methods of PERC mediated genetic engineering of TIL. FIG. 12A shows the percent (%) of pan T cells that had an indel or had CISH knocked out for both electroporation mediated genetic modification and PERC-mediated genetic modification. FIG. 12B shows the percent (%) of pan T cells that were viable at the end of the 72 hour incubation for cells treated with the non-targeting control sgRNA (NTC) or cells treated with the CISH targeting sgRNA for both electroporation mediated genetic modification and PERC-mediated genetic modification. Cells labeled as DMSO were controls that were not genetically modified. FIG. 12C shows the cell counts for pan T cells treated with the non-targeting control sgRNA (NTC) or pan T cells treated with the CISH targeting sgRNA for both electroporation mediated genetic modification and PERC-mediated genetic modification at the end of the 72 hour incubation. FIG. 12D show the cell counts of TIL edited with PERC or electroporation for each of the target genes. FIG. 12E shows the percent (%) of the TIL that had a target gene knocked out for both electroporation mediated genetic modification and PERC- mediated genetic modification.Detailed Description

[0214] Provided herein are methods for manufacturing T cells. In provided embodiments, the methods relate to genetically modifying tumor reactive TIL by gene editing followed by their expansion to generate a therapeutic TIL product.

[0215] In accordance with embodiments herein, the provided methods or processes for manufacturing T cell preparations may be useful for treating patients with a pathological disease or condition. In contrast to known methods, the methods and processes described herein can produce T cells with improved persistence, reduced exhaustion, reduced tumor mediated- immune suppression, and / or reduced reliance on cytokine administration to patients, which may lead to toxicity. Also provided herein are populations of genetically modified TIL produced by methods described herein and pharmaceutical compositions thereof.

[0216] The provided methods relate to producing a T cell therapy reactive to tumor- associated antigens. Cancer cells accumulate lots of different DNA mutations as part of the tumorigenic process. These mutations can cause amino acid changes in protein coding regions. Lor a mutation to be recognized by the immune system the protein needs to be processed intracellularly and presented on the surface with the Major Histocompatibility Complex (MHC). Peptide neoantigens (also referred to herein as neoepitopes or peptide neoepitopes) are the mutant peptides presented by the MHC complex that can be recognized by a T-cell via TCR binding. In order for the immune system to recognize the mutation, it must be expressed on the surface of the cancer cell via the MHC complex and the T cell must have a TCR that recognizes the mutated peptide. These neoantigens may be presented by MHC class I and MHC class II, and are recognized by CD8+ and CD4+ T cells respectively.

[0217] In some embodiments, the method described may be used to manufacture T cells which express cell surface receptors. The cell surface receptor may be a T cell receptor (TCR) or novel group of TCRs. In particular embodiments of the provided methods, the population of Tcells is or includes reactive T cells that express cell surface receptors, such as a T cell receptor (TCR), able to recognize peptide antigens on the surface of a target cells. Specifically, for an antigen to be recognized by the immune system the protein needs to be processed intracellularly to peptide fragments that are then presented on the surface with the Major Histocompatibility Complex (MHC). A TCR has two protein chains, which are designed to bind with specific peptides presented by a major histocompatibility complex (MHC) protein on the surface of certain cells. Since TCRs recognize peptides in the context of MHC molecules expressed on the surface of a target cell, TCRs have the potential to recognize antigens not only presented directly on the surface of target cells, e.g. cancer cells, but also presented by antigen-presenting cells, such as are present in tumor, inflammatory and infected microenvironments, and in secondary lymphoid organs. Reactive T cells expressing such cell surface receptors may be used to target and kill any target cell, including, but not limited to, infected cells, damaged cells, or dysfunctional cells. Thus, according to the embodiments described herein, the manufactured T cells expressing the cell surface receptor may be used to target and kill any target cell, including, but not limited to, infected cells, damaged cells, or dysfunctional cells. Examples of such target cells may include cancer cells, virally infected cells, bacterially infected cells, dysfunctionally activated inflammatory cells (e.g., inflammatory endothelial cells), and cells involved in dysfunctional immune reactions (e.g., cells involved in autoimmune diseases).

[0218] In some embodiments, a “T cell receptor” or “TCR” is a molecule that contains a variable a and P chains (also known as TCRa and TCRp, respectively) or a variable y and 5 chains (also known as TCRy and TCRS, respectively), or antigen-binding portions thereof, and which is capable of specifically binding to a peptide bound to an MHC molecule. In some embodiments, the TCR is in the aP form. Typically, TCRs that exist in aP and y5 forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. A TCR can be found on the surface of a T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0219] In some aspects, the reactive T cells are tumor-reactive T cells that recognize a cancer neoantigen. The majority of neoantigens arise from passenger mutations, meaning they do not infer any growth advantage to the cancer cell. A smaller number of mutations actively promote tumor growth, these are known as driver mutations. Passenger mutations are likely to give rise to neoantigens that are unique to each patient and may be present in a subset of allcancer cells. Driver mutations give rise to neoantigens that are likely to be present in all the tumor cells of an individual and potentially shared. In some embodiments of the provided method, the population of T cells contain tumor-reactive T cells that can recognize neoantigens containing passenger and / or driver mutations. In some embodiments of the provided method, the population of T cells contain tumor-infiltrating lymphocytes (TILs) which include tumor- reactive T cells.

[0220] In particular aspects, the provided methods can be used for the ex vivo production of a T cell therapy, including for the ex vivo expansion of autologous tumor-reactive T cells. In some aspects, neoantigens are ideal targets for immunotherapies because they represent diseasespecific targets. For example, such antigens generally are not present in the body before the cancer developed and are truly cancer specific, not expressed on normal cells and are not subjected to off target immune toxicity. Thus, the unique repertoire of neoantigens specific to the patient can elicit a strong immune response specific to the cancer cells, avoiding normal cells. This is an advantage over other cell therapy targets that may not be disease-specific targets, since even low levels of target antigen on normal cells can lead to severe fatal autoimmune toxicity in the context of engineered therapies that target common antigens. For example, an anti MAGE-A3-TCR program in melanoma patients was halted due to study related deaths attributed to cross reactivity with a similar target MAGE-A12, which is expressed at a low level in the brain. A significant challenge in cancer immunotherapy has been the identification of cancer targets.

[0221] Recent clinical studies have demonstrated that T cells isolated from surgically resected tumors possess TCRs that recognize neoantigens, and expanding these neoantigen reactive TIL populations and re-infusing them into the patient can in some cases result in a dramatic clinical benefit. This personalized therapy has generated remarkable clinical responses in certain patients with common epithelial tumors.

[0222] Existing methods for obtaining and generating tumor-reactive T cells are not entirely satisfactory. For example, direct isolation of tumor-reactive T cells from a subject without expansion is not feasible because therapeutically effective amounts of such cells cannot be obtained. To expand the tumor-reactive T cells, they are stimulated which may lead to a loss of reactivity. In addition, when the tumor-reactive T cells are administered to a subject, the subject is often administered one or more cytokines either at the same time as the tumor-reactive T cells and / or after administering the tumor-reactive T cells to maintain the activity of the T cells. Theone or more cytokines may cause toxicity in the subject. Tumor-reactive T cells may lack persistence in the subject being treated and tumors are immune suppressing environments, both of these facts can lead to administration of tumor-reactive T cells to not be as effective.

[0223] Moreover, TIL therapy can be limited by several mechanisms including tumor mediated-immune suppression, limited persistence, exhaustion, and toxicity associated with high dose IL-2 treatment. For instance, after receiving TIL therapy, patients are routinely treated with high dose IL2 (e.g., 720,000 lU / Kg) which in some cases is given every 8 hours for 5 days. With this approach, TIL therapy has been successful in obtaining an objective response of 43% and a complete response of 12% in patients with metastatic melanoma. However, administration with high dose IL-2 is associated with high-grade toxicities including those associated with neurological symptoms and systemic capillary leak syndrome.

[0224] Methods to produce tumor-reactive T cells with either improved effectiveness, the ability to persist, and / or reduced reliance on external cytokine administration for therapy are needed.

[0225] The provided embodiments relate to improved methods for identifying, genetically modifying, and expanding T cells ex vivo, including tumor-reactive T cells, for use in T cell therapy. In some embodiments, the provided methods improve or increase the growth and survival of T cells, such as tumor-reactive T cells, outside of the body. In particular embodiments, the methods enrich for expansion of reactive T cells compared to non-reactive T cells and promote their survival and growth in culture ex vivo. In some embodiments, the provided methods lead to increased persistence in a subject. In some embodiments, the provided methods lead to decreased administration of one or more cytokines to a subject subsequently to the administration of the tumor-reactive T cells to the subject. In some embodiments, the provided methods lead to increased tumor reactivity in the tumor-reactive T cells and / or increased cytolysis of tumor cells by the tumor-reactive T cells. In some embodiments, the resulting methods can be carried out in a closed system. The methods in some embodiments are carried out in an automated or partially automated fashion.

[0226] The provided methods contemplate that selection of cells during one or more steps of an ex vivo process for manufacturing tumor reactive T cells based on expression of CD45, CD4 or CD8, and one or more T cell activation markers associated with TIL reactivity (e.g., exhaustion marker PD-1 and / or CD39) and then genetically modifying the selected cells will result in an improved TIL therapy enriched in tumor reactive T cells with high potential for 1therapeutic efficacy for treating certain cancers. The provided methods results in a product containing tumor reactive T cells that can target many mutations and / or that contains an oligoclonal population of TCRs that are reactive to different tumor antigens, and that exhibit improved phenotype and functionality. Thus, such tumor reactive T cells offer advantages compared to existing methods in which cells are transduced to express a single neoepitope reactive TCR or in which TIL are bulk expanded from a tumor sample.

[0227] In some embodiments, the methods include, but are not limited to the steps of (1) obtaining a population of tumor-reactive TIL from a tumor sample from a subject, (2) genetically modifying the population tumor-reactive TIL by introducing a genetic disruption at a target site of a target gene (e.g, SOCS1 or CISH) in T cells of the population of tumor reactive TIL by gene editing to produce a population of genetically modified tumor-reactive TIL, and (3) performing an expansion by culturing the population of genetically modified TILs by incubation or culture with one or more T-cell stimulating agents of lymphocytes to produce a population of genetically modified and expanded TIL, and (4) harvesting the population of genetically modified and expanded tumor-reactive TIL.

[0228] In some embodiments, prior to genetically modifying the TIL the methods can include a first expansion of tumor-reactive TIL by culture with one or more first T-cell stimulating agents. In some embodiments, the methods include, but are not limited to the steps of (1) obtaining a population of tumor-reactive TIL from a tumor sample from a subject, (2) performing an expansion by culturing the population of tumor-reactive TIL by incubation or culture with one or more first T-cell stimulating agents of lymphocytes to produce a first population of expanded tumor-reactive TIL, (3) genetically modifying the first population of expanded tumor-reactive TIL by introducing a genetic disruption at a target site of a target gene (e.g, SOCS1 or CISH) in T cells of the population of tumor reactive TIL by gene editing to produce a population of genetically modified tumor-reactive TIL, and (4) performing a second expansion by culturing the population of genetically modified TILs by incubation or culture with one or more second T-cell stimulating agents of lymphocytes to produce a population of genetically modified and expanded TIL, and (5) harvesting the population of genetically modified and expanded tumor-reactive TIL. In some embodiments, the expansion can be carried out by methods to achieve rapid expansion (REP) of TILs.

[0229] In some embodiments, the methods include two expansions after genetically engineering the TIL. In some embodiments, the methods include, but are not limited to the stepsof (1) obtaining a population of tumor-reactive TIL from a tumor sample from a subject, (2) genetically modifying the population tumor-reactive TIL by introducing a genetic disruption at a target site of a target gene (e.g, SOCS1 or CISH) in T cells of the population of tumor reactive TIL by gene editing to produce a population of genetically modified tumor-reactive TIL, (3) performing a first expansion by culturing the population of genetically modified TILs by incubation or culturing with one or more first T-cell stimulating agents of lymphocytes to produce a first population of genetically modified and expanded TIL, (4) performing a second expansion by culturing the first population of genetically modified and expanded TIL by incubation or culture with one or more second T-cell stimulating agents of lymphocytes to produce a second population of genetically modified and expanded TIL, and (5) harvesting the population of genetically modified and expanded tumor-reactive TIL.

[0230] In some embodiments, prior to introducing a genetic disruption, the method includes one or more activations. In some embodiments, the methods include, but are not limited to the steps of (1) obtaining a population of tumor-reactive TIL from a tumor sample from a subject, (2) activating the population of tumor-reactive TIL by incubating with a T cell activation reagent to create an activated population, (3) genetically modifying the activated population by introducing a genetic disruption at a target site of a target gene (e.g, SOCS1 or CISH) in T cells of the activated population by gene editing to produce a population of genetically modified tumor-reactive TIL, and (4) performing an expansion by culturing the population of genetically modified TILs by incubation or culture with one or more T-cell stimulating agents of lymphocytes to produce a population of genetically modified and expanded TIL, and (5) harvesting the population of genetically modified and expanded tumor-reactive TIL. In some embodiments, the methods include, but are not limited to the steps of (1) obtaining a population of tumor-reactive TIL from a tumor sample from a subject, (2) activating the population of tumor-reactive TIL by incubating with a first T cell activation reagent to produce a first activated population, (3) further activating the first activated population by incubating with a second T cell activation reagent to create a second activated population (4) genetically modifying the second activated population by introducing a genetic disruption at a target site of a target gene (e.g, SOCS1 or CISH) in T cells of the second activated population by gene editing to produce a population of genetically modified tumor-reactive TIL, and (5) performing an expansion by culturing the population of genetically modified TILs by incubation or culture with one or more T-cell stimulating agents of lymphocytes to produce a population of geneticallymodified and expanded TIL, and (6) harvesting the population of genetically modified and expanded tumor-reactive TIL.

[0231] In some embodiments, the population of tumor-reactive TIL is obtained from a tumor sample from a subject by selecting from an input population of cells containing T lymphocytes obtained from a donor subject, cells positive for one or more T cell activation marker. Hence, in some embodiments, the tumor-reactive TIL are selected TIL obtained from a tumor sample. In some embodiments, a tumor sample is digested and / or homogenized to produce a suspension and the input population of cells are the suspension of tumor cells. In some embodiments, the one or more T cell activation markers are markers that are able to enrich for tumor-reactive TIL from the tumor. Exemplary T cell activation markers are described herein. In some embodiments, the T cell activation markers include PD-1 and CD39. In some embodiments, tumor-reactive TIL are obtained from the tumor by selecting for T cells surface positive for PD- 1 and CD39. In some embodiments, cells surface positive for one or more T cell activation markers are selected from the input population of cells (tumor cell suspension) by contacting the cells with one or more antibodies directed against the T cell activation markers and isolating the subset of cells positive for the T cell activation markers. In some embodiments, selecting tumor- reactive cells is carried out by flow cytometry-based cell sorting.

[0232] PD-1 and CD39 are each checkpoint molecules that also can represent markers of exhausted T cells. They also are markers that are activation markers or upregulation markers in that their expression is increased upon tumor reactivity, which is a natural mechanism of immune suppression of the host immune response. For instance, the immune system is designed to shut itself off to avoid an overactive immune response in order to avoid inflammatory and autoimmune responses. In this way, an immune response is initially developed against cancer but this can be thwarted by the upregulation of certain checkpoint molecules, like PD-1 andCD39, that can inhibit the immune response. As these are markers that are upregulated on cells in which an immune response is being developed, it is contemplated by the provided methods that such markers serve as powerful markers for specifically enriching for tumor reactive T. By specifically selecting for tumor reactive cells based on these activation markers, the provided methods avoid bulk expansion of T cells from a tumor source that would include a number of bystander cells that are not tumor reactive or that could exhibit inhibitory activity, such as Tregs.

[0233] In provided embodiments, following selection for tumor reactivity, TIL derived from tumor-bearing subjects are subjected to gene editing by CRISPR-Cas mediated editing of the a target locus (e.g., SOCS1) followed by expansion (e.g. rapid expansion) as described. Various exemplary target loci are described and can be edited in accord with provided methods. In some embodiments, the genetic disruptions result in gene deletion to knockout the target locus in the cell. In some embodiments, the target locus is a target site in SOCS1 resulting in genetic disruption (e.g. deletion) and knockout of SOCS1 in the cells. In some embodiments, the methods provided herein to genetically modify T cells enriched in tumor-reactive TIL followed by their expansion provides a strategy for enhancing TIL phenotype and function. For instance, results herein demonstrate that genetic modification of target loci in selected tumor-reactive TIL have increased polyfunctionality in response to polyclonal stimulation, increased function as determined by cytotoxicity proliferation and / or IFN-gamma secretion against autologous tumor cells.

[0234] In some embodiments, the cells are genetically modified by genetic disruption of a target site in SOCS1. SOCS1 is a gene expressed in response to T cell receptor or cytokine receptor signaling. SOCS1 encodes a protein which mediates JAK / STAT degradation, thereby suppressing T cell receptor signaling. In some embodiments, reduced expression or genetic deletion of SOCS1 in tumor reactive T cells increases control of tumor growth, cytokine secretion, and / or responsiveness to IL- 12 in the tumor reactive T cells.

[0235] In some embodiments, the cells are genetically modified by genetic disruption of a target site in CISH. CISH is a gene induced by T cell receptor stimulation. The protein encoded by CISH interacts with PLC-yl and causes degradation of the TCR signaling intermediate. In some embodiments, reduced expression or genetic deletion CISH in tumor reactive T cells enhances CD8+ T cell expansion, cytokine polyfunctionality and increases tumor killing.

[0236] In some embodiments, expression of markers PD-1 and / or CD39, such as PD-1 and CD39, are used to enrich TIL immediately after tumor dissociation, either at the endpoint of tumor fragment culture or immediately after mechanical / enzymatic creation of a single cell suspension from tumor fragments. In some embodiments, PD-1 and / or CD39 expressing cells, among cells that also express CD45 and CD4 or CD8, are isolated from either tumor fragment cultures or single cell suspensions generated through enzymatic digestion.

[0237] In aspects of the provided methods, after selection and prior to genetic modification by gene editing, selected cells can be expanded (e.g., first expansion) in the presence of one ormore T cell stimulating agent. In aspects of the provided methods, after genetic modification by gene editing, genetically modified cells can be expanded (e.g., second expansion) in the presence of one or more T cell stimulating agent. In some embodiments, the T cell stimulating agent can include any one or more recombinant cytokines IL-2, IL-7, IL- 15, IL-21, IL-25, IL-23, IL-27 or IL-25, such as generally at least one or more of IL-2, , IL-7, IL- 15 and / or IL-21. In some embodiments, the T cell stimulating agent can include recombinant IL-2. In some embodiments, the T cell stimulating agent can include IL- 15 and IL-21. In some embodiments, the T cell stimulating agent can include IL15 and IL-7. In some embodiments, the T cell stimulating agent can further include an anti-CD3 antibody (e.g. OKT3). In some embodiments, the T cell stimulating agent can further include non-dividing autologous peripheral blood mononuclear cells (PBMCs), such as autologous irradiated PBMCs. In some embodiments, the expansion, such as the first expansion and / or second expansion is carried out in the presence of one or more recombinant cytokine (e.g, IL-2 or IL-15 and IL-21), anti-CD3(OKT3) and autologous irradiated PBMCs.

[0238] In an exemplary method, tumor cells are digested and selected by Fluorescence Activator Sorting for CD4, CD8, PD1 and CD39 positivity. Following sorting, selected TIL are put through one round of Rapid expansion (REP) by culture with one or more T cell stimulating agent (e.g., by stimulating with 30ng / ml of OKT3, feeder cells (200:1), and 3000 lU / ml of IL2), genetically modified by CRISPR-Cas-mediated gene editing to genetically disrupt a target loci (e.g., SOCS1), and subsequently the genetically modified cells are put through a second round of REP expansion by culture with one or more T cell stimulating agent (e.g., by stimulating with 30ng / ml of OKT3, feeder cells (200:1), and 3000 lU / ml of IL2). In some embodiments, the step of genetically modifying the cells by CRISPR-Cas mediated gene editing includes activating cells (e.g., expanded tumor-reactive TIL) with an anti-CD3 / anti-CD28 agonist and within 3 days (e.g., 72 hours) contacting the cells with sgRNA targeting specific genes of interest in combination with a CAS (e.g., CAS9), such as delivered to TIL in the form of ribonucleotide particles at a 2: 1 ratio of sgRNA to CAS9 using electroporation. After rest following the electroporation (e.g., rest for about 3 hours) the genetically modified cells are then subjected to the expansion.

[0239] In particular embodiments, the genetic modification by gene editing (e.g., CRISPR- CAS-mediated genomic engineering) is performed on patient-derived TIL selected for tumor reactivity, such as based on PD1 and CD39 positivity. As compared to existing methods, usingthis approach can increase the efficacy of TIL populations that have been enriched for reactivity by targeting genes whose deletion enhances cytotoxicity, proliferation, phenotype, resistance to the tumor microenvironment and decreases IL2 dependency. Results herein have demonstrated successful knock out of PDCD1 (PD-1), MAPK14, RASA2, CISH, and SOCS1 in directly selected TIL.

[0240] Among provided embodiments herein are the surprising results that gene editing to enhance the phenotype and function of patient-derived TIL enriched for tumor reactivity may support TIL therapy without cytokine support, such as without IL2. Using a CRISPR-CAS mediated approach, results herein show that knockout (KO) of SOCS1 enhances TIL cytolysis of tumor target cells in the absence of IL2. Furthermore, results herein demonstrate that SOCS1 KO PD1+CD39+selected TIL is sensitive to IL2 signaling as the percentage of cells with SOCS1 knocked out decreases at the end rapid expansion with IL2 but is stable when expanded in the presence of IL15 / IL21. Collectively, this data suggests that SOCS1 KO decreases TIL dependency on IL2 signaling. When translated into the clinic, patients treated with selected SOCS1 KO TIL, such as generated and expanded in accord with the provided embodiments, could potentially not need IL2 support which would decrease the overall toxicity associated with TIL treatment.

[0241] In embodiments of the provided methods, the population of T cells is obtained from a biological sample known to contain T cells. In some embodiments, the population of T cells is enriched from a biological sample from a subject, in particular a human subject. The biological sample can be any sample containing a bulk population of T cells. In some embodiments, the biological sample is or includes peripheral blood mononuclear cells. In some embodiments, the biological sample is a peripheral blood or serum sample. In some embodiments, the biological sample is a lymph node sample. In some embodiments, the biological sample is a tumor sample. In some aspects, the bulk T cells can include tumor- infiltrating T cells (TILs). In some embodiments, the subject is a human subject. In some embodiments the subject is a subject having a cancer, viral infection, bacterial infection, or is a subject with an inflammatory condition. In particular embodiments, the subject has a cancer.

[0242] In aspects of the provided methods, the starting source of cells (input sample) in the method can be tumor fragments (e.g. 1-8 mm diameter fragments) or can be a single cell suspension preparation from enzymatic digestion of tumor fragments. While certain sources may be superior for some tumor types, both fragments and single cell suspensions can support Tcell expansion and enrichment of tumor-reactive T cells. In some cases, the tumor cell source can be chosen depending on the tumor type or cancer, such as to optimize or increase expansion and enrichment of tumor-reactive T cells from the tumor. In one example, the cancer is a melanoma and the starting population of lymphocytes are tumor fragments, such as from a resected tumor. In another example, the cancer is a colorectal cancer and the starting population of lymphocytes is a single cell suspension obtained by enzymatic digestion, e.g. collagenase and / or hyaluronidase, of tumor fragments.

[0243] In some embodiments, the methods produce or expand T cells for use in adoptive cell therapy for treating a disease or condition in which cells or tissue associated with the disease or condition is known or suspected of expressing an antigen target recognized by the T cells. In some embodiments, the T cell therapy is autologous to the subject. In some embodiments, the T cell therapy is allogeneic to the subject.

[0244] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0245] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. EX VIVO METHODS OF PRODUCING TIL COMPOSITIONS ENRICHED IN TUMOR-REACTIVE T CELLS

[0246] Various embodiments of the provided methods involve the ex vivo expansion genetic modification of patient-derived tumor reactive T cells for the production of a TIL therapeutic composition, particularly for use in connection with treating cancer. In some embodiments, the method of manufacturing involves the growth and manipulation of patient cells outside of the body.

[0247] In some embodiments, the methods are for generating genetically modified tumor- reactive tumor infiltrating lymphocytes (TILs), in which the method includes (a) obtaining a population of tumor-reactive TIL from a tumor sample from a subject; (b) introducing a genetic disruption at a target site within a target gene selected from the group consisting of SOCSLCISH, PDCD1 (PD-1), TIGIT, RC3H1 (Roquin), CM1P (gene encoding C-Maf Inducing Protein), CD39, LAG3, TIM3, MAPK14, and RASA2 (gene encoding RAS p21 protein activator 2) in T cells of the population of tumor-reactive TIL by gene editing to produce a population of genetically modified tumor-reactive TIL; (c) performing an expansion by culture of the population of genetically modified tumor-reactive TIL with one or more T-cell stimulating agent to produce a population of genetically modified and expanded tumor-reactive TIL; and (d) harvesting the population of genetically modified and expanded tumor-reactive TIL from step (c). In some embodiments, the population of tumor-reactive TIL from a tumor sample from a subject can be processed by digestion of tumor fragments to create a single cell suspension (e.g. population).

[0248] The provided embodiments relate to processes for preparing a therapeutic TIL composition enriched for tumor reactive T cells that involves a direct selection of cells to yield a tumor reactive cell population that is further expanded. In the provided methods, a TIL tumor sample is obtained that contains or is expected to contain tumor reactive T cells (e.g. first population). In one embodiment, this population can be processed by digestion to create a single cell suspension (e.g. second population). In some embodiments, this second population is sorted to select for cells that are enriched for tumor reactive T cells and to minimize the presence of bystander cells, such as regulatory T cells. In some embodiments, the selection is for cells positive for PD-1 and / or CD39. In some embodiments, the selection is for cells positive for PD- 1 and CD39. In provided methods, the T cells are sorted directly after tumor digest or after a short period of cell culture for cells surface positive for CD39, and PD1 to create a population of selected T cells. This process removes the nonreactive and inhibitory ‘bystander’ cells, resulting in T cell product enriched in neoantigen reactive T cells. The selection yields a selected or sorted population of cells (e.g. in some cases also called a third population), which then can be expanded to create a therapeutic composition containing an expanded population of tumor specific reactive cells (e.g. in some cases also called a fourth population).

[0249] In some embodiments, provided methods relate to a method of generating genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), in which the method includes (a) selecting cells surface positive for one or more T cell activation markers from a tumor sample from a subject to produce a population of tumor-reactive TIL; and (b) introducing a genetic disruption at a target site within a target gene selected from the group consisting of SOCSL CISH, PDCD1 (PD-1), TIGIT, RC3H1 (Roquin), CMIP (gene encoding C-Maf InducingProtein), CD39, LAG3, TIM3, MAPK14, and RASA2 (gene encoding RAS p21 protein activator 2) in T cells of the population of tumor-reactive TIL by gene editing to produce a population of genetically modified tumor-reactive TIL; (c) performing an expansion by culture of the population of genetically modified tumor-reactive TIL with one or more T-cell stimulating agent to produce a population of genetically modified and expanded tumor-reactive TIL; and (d) harvesting the population of genetically modified and expanded tumor-reactive TIL from step (c). The one or more T cell activation markers can be any as described herein. In some embodiments, the one or more T cell activation markers are PD-1 and / or CD39. In some embodiments, the one or more T cell activation markers are PD-1 and CD39.

[0250] In some embodiments, provided methods of generating genetically modified tumor- reactive tumor infiltrating lymphocytes (TILs) using any of the steps described in subsequent sections. Exemplary methods for producing genetically modified tumor-reactive infiltrating lymphocytes (TILs) are shown below in Table 1.Table 1: Exemplary methods of generating genetically engineered TIL

[0251] In some embodiments, cells are then expanded into clinically relevant numbers of tumor specific T cells. In aspects of the provided methods, the T cells from the selected population (third population), such as present from the sorted T selected cells from a resected tumor fragment or a single cell suspension therefrom, are expanded by culture in the presence of one or more T cell stimulatory agent(s) under conditions for stimulating the T cells. In some embodiments, the final expanded therapeutic composition is formulated with a cyroprotectant for cryopreservation.A. Sample Containing T cells

[0252] The provided methods include selecting or obtaining an input sample of T cells from a biological sample, which can be used as the source or input of T cells for stimulation with one or more T cell stimulatory agents(s) (e.g. recombinant IL-2 or other T cell stimulating cytokines and / or anti-CD3). In some embodiments, the T cells are from a biological sample from a subject that is known or likely to contain tumor reactive T cells. In some embodiments, the biological sample provides an input population of cells that is a single cell suspension (SCS), which can be used for subsequent selection of TILs for expansion as described in Section II.B and II.C. In some embodiments, the biological sample is processed to provide an input population of cells that is a single cell suspension, which can be used for subsequent selection of TILs for expansion as described in Section II.B and II.C. For instance, particular methods include processing tumor fragments by dissociation using homogenization and / or enzymatic methods and filtering the dissociated cells to prepare a single cell suspension as an input population of cells.

[0253] In aspects of any of the provided embodiments, a suitable biological sample from a subject, such as from a patient of interest, i.e., a patient suspected of having or known to have cancer, is obtained. In some embodiments, the sample is one that is known or suspected of containing T cells, such as T cells that may be or may likely express an endogenous T cell receptor (TCR). The biological sample may be derived from any initial source that would contain or is suspected of containing such T cells. In some aspects, biological sample sources of interest include, but are not limited to, many different physiological sources, e.g. tissue derived samples, e.g. homogenates, and blood or derivatives thereof.

[0254] Any of a variety of biological samples can be used as a source of potentially reactive T cells. Although the tumor and downstream lymph nodes may have the highest frequency of reactive T cells (Powell et al., Clin. Cancer. Res., 2014), other sample sources also can be used.In some cases the sample is a tumor sample, a tertiary lymphoid site, a draining lymph node, peripheral blood or bone marrow. In some embodiments, the biological sample is a tumor sample. In some embodiments, the biological sample is a lymph sample. In some embodiments, the biological sample is a peripheral blood sample.

[0255] The biological samples include tissue, fluid, and other samples taken directly from the subject to obtain an input sample, or can undergo one or more processing steps, such as separation, e.g. selection or enrichment, centrifugation, washing, and / or incubation, to obtain or produce an input sample. The input sample containing T cells can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, and tumor samples, including processed samples derived therefrom.

[0256] In some aspects, the sample is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0257] In many embodiments, the sample may be derived from fluids in which the T cells of interest are at least suspected of being present. In many embodiments, a suitable initial source for the sample is blood. In some embodiments, the biological sample is a blood-derived sample. The blood-derived sample may be derived from whole blood or a fraction thereof, e.g. serum, plasma, etc., where in many embodiments the sample is derived from blood cells harvested from whole blood. In some aspects, the sample source contains mononuclear cells. For example, a biological sample is or contains peripheral blood mononuclear cells (PBMCs) or is derived from PBMCs.

[0258] In some embodiments in which the sample is a PBMC derived sample, the sample is generally a fluid PBMC derived sample. Any convenient methodology for producing a fluid PBMC sample may be employed. In many embodiments, the fluid PBMC derived sample is prepared by separating PBMCs from whole blood, i.e., collecting PBMCs, e.g., bycentrifugation (such as by Ficoll-Hypaque density gradient centrifugation, where representative protocols for such separation procedures are disclosed in WO 98 / 15646 and U.S. Pat. No. 5,985,565).

[0259] In some embodiments, the sample is a tumor sample and thereby provides a source of tumor- infiltrating lymphocytes (TILs). In some aspects, TILs are T cells that have left the bloodstream of a subject and migrated into or infiltrated a tumor. In particular aspects, TILs are reactive to a tumor antigen.

[0260] A patient tumor sample may be obtained by any of a variety of methods in which the method obtains a sample that contains a mixture of tumor and TIL cells. In some embodiments, the tumor sample is obtained by surgical resection. In some embodiments, the tumor sample is obtained by needle biopsy. In general, the tumor sample may be from any solid tumor, including primary tumors, invasive tumors or metastatic tumors. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematological malignancy.

[0261] In some embodiments, the solid tumor may be of any cancer type, including, but not limited to, ovarian, vulva, endometrial, urothelial, breast, pancreatic, prostate, colorectal, lung, brain, renal, stomach (gastrointestinal), and skin (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the tumor is from a patient with a cancer, including, but not limited to, ovarian, vulva, endometrial, urothelial, breast, colorectal, lung, renal, and skin (including but not limited to melanoma). In some embodiments, the tumor is from a patient with an ovarian cancer. In some embodiments, the tumor is from a patient with cancer of the vulvar. In some embodiments, the tumor is from a patient with an endometrial cancer. In some embodiments, the tumor is from a patient with a urothelial cancer. In some embodiments, the tumor is from a patient with a breast cancer. In some embodiments, the tumor is from a patient with a colorectal cancer. In some embodiments, the tumor is from a patient with a lung cancer. In some embodiments, the tumor is from a patient with a renal cancer. In some embodiments, the tumor is from a patient with melanoma. In particular embodiments, the tumor is from a patient to be treated as described in Section III.

[0262] In particular embodiments, a T cell population is one that includes both CD4+ and CD8+ T cells. Many cancers, including solid tumors, such as many common epithelial indications (e.g. GI), express class I and class II restricted mutations. In order for a T cell product to target such indications, e.g. common epithelial indications, it is contemplated thatboth CD8+ T cells to recognize class I MHC-restricted molecules and CD4+ T cells to recognize Class II MHC-restricted molecules are necessary.

[0263] The sample may be obtained from a variety of different subjects / patients / hosts. Generally such hosts are “mammals” or “mammalian,” where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the hosts will be humans.

[0264] In some aspects, the subject is a human. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. In some embodiments, the sample is autologous to a subject to be treated, such as a subject who is a patient in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or expanded in accord with the provided methods. In some embodiments, the sample is allogenic to a subject to be treated.

[0265] In provided embodiments, the obtained tumor sample is fragmented into small pieces of between at or about 1 mm3and at or about 8 mm3in size, such as between at or about 1 mm3and at or about 3 mm3, between at or about 1 mm3and at or about 4 mm3, between at or about 1 mm3and at or about 2 mm3. In some embodiments, the tumor fragment is from about -3 mm3. In some embodiments, the tumor fragment is from about 1-3 mm3. In some embodiments, the tumor fragment is obtained by physical fragmentation, such as by dissection. In some embodiments, the tumor fragment is obtained by sharp dissection.

[0266] In some of any of the provided embodiments, the obtained tumor sample is fragmented into small pieces of between at or about 1 mm and at or about 8 mm in diameter, such as between at or about 1 mm and at or about 6 mm in diameter, between at or about 1 mm and at or about 4 mm in diameter, between at or about 1 mm and at or about 2 mm in diameter. In some embodiments, the tumor fragment is from about 3 mm in diameter. In some embodiments, the tumor fragment is from about 1-2 mm in diameter. In some embodiments, the tumor fragment is obtained by physical fragmentation, such as by dissection. In some embodiments, the tumor fragment is obtained by sharp dissection.

[0267] In some embodiments, the tumor sample is cryopreserved prior to fragmentation. In some embodiments, the tumor fragments are cryopreserved.

[0268] In some embodiments, tumor fragments are used as a source to prepare a single cell suspension for use as an input sample of T cells in the provided methods. In someembodiments, the provided methods involve obtaining cells from the tumor fragments, such as by enzymatic digestion of tumor fragments to obtain TILs. Enzymatic digestion can be carried out, in part, using a collagenase, such as a type IV collagenase or a type VII collagenase. Collagenase is an enzyme that degrades the collagen network embedded in the extracellular matrix of the cell (Eikenes et al. Anticancer Research, 2010). The enzyme, such as a collagenase, can be present in media for the enzymatic digestion at a concentration of from at or about 1 mg / mL to at or about 5 mg / mL, such as at or about 1 mg / mL, at or about 2 mg / mL, at or about 3 mg / mL, at or about 4 mg / mL at or about 5 mg / mL, at or about 6 mg / mL, at or about 7 mg / mL, at or about 8 mg / mL or at or about 9 mg / mL, at or about 10 mg / mL or any value between any of the foregoing. In some embodiments, collagenase is present in the media at a concentration of from about 5 mg / mL to about 10 mg / mL. In some embodiments, the concentration is about 5 mg / mL. In some embodiments, the concentration is 10 mg / mL. In some embodiments, the collagenase is a type IV collagenase. In some embodiments, the collagenase is a type VII collagenase. In some embodiments, the enzymatic digestion is with a media that includes type IV collagenase, such as from at or about 5 mg / mL to at or about 10 mg / mL. In some embodiments, the enzymatic digestion is with a media that includes type VII collagenase, such as from at or about 5 mg / mL to at or about 10 mg / mL. In some embodiments, if a more gentle digestion is desired at or about 5 mg / mL collagenase is used. In some embodiments, if a more complete digestion is desired a higher concentration of collagenase is used, such as at or abut 10 mg / mL collagenase. In some embodiments, the collagenase is a type IV collagenase. In some embodiments, the collagenase is a type VII collagenase.

[0269] In some embodiments, enzymatic digestion can be carried out, in part, using a hyaluronidase. Hyaluronidase is a hyaluronic acid-metabolizing enzyme, subsequently enhancing cell membrane permeability (Eikenes et al. Anticancer Research, 2010). The enzyme, such as a hyaluronidase, can be present in media for the enzymatic digestion at a concentration of from at or about 5 mg / mL to at or about 10 mg / mL, such as at or about 5 mg / mL, at or about 6 mg / mL, at or about 7 mg / mL, at or about 8 mg / mL or at or about 9 mg / mL, at or about 10 mg / mL or any value between any of the foregoing. In some embodiments, the enzymatic digestion is with a media that includes type II hyaluronidase, such as from at or about 5 mg / mL to at or about 10 mg / mL. In some embodiments, if a more gentle digestion is desired at or about 5 mg / mL hyaluronidase is used. In some embodiments, if a morecomplete digestion is desired a higher concentration of hyaluronidase is used, such as at or abut 10 mg / mL hyaluronidase.

[0270] In some embodiments, DNase is also present in the media for during the enzymatic digestion. DNase is an enzyme that degrades any free DNA released into the media as a result of the tumor fragment digestion process. The enzyme, such as a DNase I, can be present in media for the enzymatic digestion at a concentration of from at or about 5,000 units / mL to at or about 10,000 units / mL, such as at or about 5,000 units / mL, at or about 6,000 units / mL, at or about 7,000 units / mL, at or about 8,000 units / mL or at or about 9,000 units / mL, at or about 10,000 units / mL or any value between any of the foregoing. In some embodiments, the enzymatic digestion is with a media that includes DNase I, such as from at or about 5,000 units / mL to at or about 10,000 units / mL.

[0271] In some embodiments, enzymes from the Miltenyi human tumor dissociation kit can be used (e.g. Cat. O. 130-095-929; Miltenyi Biotec). The enzymatic media containing the enzyme can be a serum-free media, such as any as described. In particular embodiments, enzymatic media includes hyaluronidase and / or collagenase, e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate (e.g. GlutaMAX), 10 mg / mL gentamicin, 10,000 units / mL of DNase I, 10 mg / mL of collagenase and 10 mg / mL of hyaluronidase).

[0272] The tumor fragment is then mechanically dissected to dissociate the TILs, e.g., using a tissue dissociator. An example of a tissue dissociator is GentleMACs™ (Miltenyi Biotec) to homogenize the tissue. Tumor digests may be produced by placing the tumor in enzymatic media and mechanically dissociating the tumor for approximately 1 minute, followed by incubation for 30 minutes at 37 °C in 5% CO2, followed by repeated cycles of mechanical dissociation and incubation under the foregoing conditions until only small tissue pieces are present. In some embodiments, tumor digests are subjected to homogenization and enzymatic digestion by incubation in the enzyme cocktail for 15 minutes to 2 hours, such as for at or about 30 minutes to 60 minutes. In some embodiments, tumor digests are subjected to homogenization and enzymatic digestion by incubation in the enzyme cocktail for about 60 minutes. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using FICOLL can be performed to remove these cells. In some embodiments, a single cell suspension is prepared following processing of the tumor fragments by straining the cells through a filter to remove debris, such as a 70 pm strainer. In some cases, separation can be achieved by centrifugation, in which case the cellpellet can be resuspended and strained through a e.g. 70 pm strainer to remove debris. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133 Al, the disclosure of which is incorporated by reference herein. Any of the foregoing methods may be used in any of the embodiments described herein for methods of obtaining TILs for use in the provided methods.

[0273] In some embodiments, a single cell suspension for use as an input sample comprises from at or about 1 x 106dissociated tumor cells to at or about 1000 x 106dissociated tumor cells, such as 1 x 106to 500 x 106dissociated tumor cells, 1 x 106to 100 x 106dissociated tumor cells, 1 x 106to 50 x 106dissociated tumor cells, 1 x 106to 10 x 106dissociated tumor cells, 10 x 106to 1000 x 106dissociated tumor cells, 10 x 106to 100 x 106dissociated tumor cells, 10 x 106to 500 x 106dissociated tumor cells, 10 x 106to 50 x 106dissociated tumor cells, 50 x 106to 1000 x 106dissociated tumor cells, 50 x 106to 500 x 106dissociated tumor cells, 50 x 106to 100 x 106dissociated tumor cells, 100 x 106to 1000 x 106dissociated tumor cells, 100 x 106to 500 x 106dissociated tumor cells, or 500 x 106to 1000 x 106dissociated tumor cells. In some embodiments, a single cell suspension for use as an input sample of T cells comprises from at or about or at least at or about 10 x 106dissociated tumor cells , 20 x 106dissociated tumor cells, 30 x 106dissociated tumor cells, 40 x 106dissociated tumor cells, 50 x 106dissociated tumor cells, 60 x 106dissociated tumor cells, 70 x 106dissociated tumor cells, 80 x 106dissociated tumor cells, 90 x 106dissociated tumor cells, or 100 x 106dissociated tumor cells In some embodiments, a single cell suspension for use as an input sample of T cells comprises from at or about 10 x 106dissociated tumor cells to at or about 100 x 106dissociated tumor cells.B. Selection of Cells

[0274] In embodiments of the provided methods, the methods involve selecting or enriching from the first population of T cells (e.g. dissociated tumor cells), cells that are likely or suspected of being tumor reactive T cells by selecting or isolating T cells that are surface positive for one or more T cell activation marker. In some embodiments, the one or more T cell activation markers are selected from the group consisting of PD-1, CD39, TIGIT, CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (0X40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM-3 and LAG-3. In some embodiments, the one or more T cell activation markers are the exhaustion markers PD-1 and CD39. In some embodiments, T cells that arepositive for one or more T cell activation markers (e.g., PD-1 and CD39) are selected or enriched from a first population of T cells that have been obtained from a biological sample, such as described in Section LA. In particular embodiments, the selection is from cells present in the single cell suspension dissociated tumor cell sample. In some embodiments, the enriched or selected population of cells is used in subsequent processing steps, such as subsequent processing steps involving expansion in accord with the provided methods.

[0275] Methods for selection of surface receptors on cells can be by any of a number of techniques, such as generally involving antibody binding with an antibody specific reagent and subsequent enrichment by magnetic separation or fluorescence-activated cell sorting (FACS).

[0276] In some embodiments, cells are selected directly from a single cell suspension input sample prepared by enzymatic or mechanical digestion of tumor fragments, in which the selection is carried out by positive selection (e.g., CD39 / PD1 positive selection). In some embodiments, the selected cells are then stimulated for expansion using methods as described, such as by incubation or culture in the presence of one or more of IL-2, IL-7, IL- 15 or IL-21. In some embodiments, the stimulation would not include culture with an anti-CD3 antibody (OKT3) or other costimulatory molecules. In some embodiments, the stimulation may include culture with an anti-CD3 antibody (OKT3) or other costimulatory molecules.

[0277] In some embodiments, selection is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (marker111811) on the positively or negatively selected cells, respectively.

[0278] In particular embodiments, a T cell population is one that includes both CD4+ and CD8+ T cells. Many cancers, including solid tumors, such as many common epithelial indications (e.g. GI), express class I and class II restricted mutations. In order for a T cell product to target such indications, e.g. common epithelial indications, it is contemplated that both CD8+ T cells to recognize class I MHC-restricted molecules and CD4+ T cells to recognize Class II MHC-restricted molecules are necessary.

[0279] In some embodiments, the methods include selection of the one or more T cell activation markers (e.g., PD-1+ and CD39+ cells) from among a lymphocyte subject of cells that includes CD4+ and CD8+ T cells. In one method, lymphocytes can be selected by positiveselection for CD3+ cells. In another method, lymphocytes can be selected by positive selection for CD4+ and / or CD8+ cells. In some aspects, a CD4+or CD8+selection step, such as positive selection for CD4 and positive selection for CD8, is used to separate CD4+helper and CD8+cytotoxic T cells. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some embodiments, the methods include enriching for CD4+ and CD8+ T cells by selecting for T cells surface positive for CD3 or by sequential or simultaneous selection for T cells surface positive for CD4 and T cells surface positive for CD8. Such CD3+ T cells, or CD4+and / or CD8+populations, can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on tumor-reactive T cells or on T cells having expression of T cell markers associated with tumor-reactive T cells, e.g. as described above.

[0280] In some embodiments, the selections produces an enriched population of cells, such as a population of cells enriched for CD3+ T cells or CD4+ cells and CD8+ cells, that are further positive for CD39 and one or more the one or more T cell activation markers are selected from the group consisting of PD-1, TIGIT, CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (0X40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM-3 and LAG-3. In some embodiments, such cells include or are enriched for tumor-reactive T cells or T cells associated with tumor-reactive T cells.

[0281] In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or twenty-one of the T cell activation markers selected from the group consisting of PD-1, TIGIT, CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (0X40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM-3 and LAG-3. In some embodiments, such cells include or are enriched for tumor-reactive T cells or T cells associated with tumor-reactive T cells.

[0282] In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and one or more the one or more T cell activation markers are selected from the group consisting of PD-1, TIGIT, CD107a, CD103, CD137 (4- 1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (0X40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM-3 and LAG-3. In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and PD-1. In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and TIGIT. In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CD107a. In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CD103. In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CD137 (4-1BB). In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CD59. In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CD90. In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CD36. In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CD38. In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CD30. In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CD154 (CD40L). In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CD134 (0X40). In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CD 152 (CTLA-4). In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CD160. In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CXCR5 (CD195). In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CD244. In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CD258 (LIGHT). In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CD256 (APRIL). In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and CD272 (BTLA-4). In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and TIM-3. In some embodiments, the selection produces an enriched population of cells, such as a population of cell enriched for CD39 and LAG-3.

[0283] In some embodiments, the selections produces an enriched population of cells, such as a population of cells enriched for CD3+ T cells or CD4+ cells and CD8+ cells, that are further positive for PD-1 and CD39. In some embodiments, such cells include or are enriched for tumor-reactive T cells or T cells associated with tumor-reactive T cells.

[0284] In some embodiments, enriching for a T cell that is surface positive for one or more cell surface marker includes any method for separation based on such markers. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. Methods of selection of cells include, but are not limited to, bead selection (e.g. serial bead passage for positive / negative selection of cells), immunoaffinity chromatography (e.g. serial elution for positive / negative selection, and flow cytometry sorting. For use in accord with the provided methods, the selection method meets GMP standards.

[0285] In some embodiments, the provided methods relate to method of generating genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), in which the method includes:(a) contacting cells from a tumor sample from a subject with a binding agent(s) that detects one or more T cell activation markers; (b) selecting cells from step (a) positive for the one or more T cell activation markers to produce a population of tumor-reactive TIL; (c) introducing a genetic disruption at a target site within a target gene selected from the group consisting of SOCS1, GISH. PDCD1 (PD-1), TIGIT, RC3H1 (Roquin), CMIP (gene encoding C-Maf Inducing Protein), CD39, LAG3, TIM3, MAPK14, and RASA2 (gene encoding RAS p21 protein activator 2) in T cells of the population of tumor-reactive TIL by gene editing to produce a population of genetically modified tumor-reactive TIL; (d) performing an expansion by culture of the population of genetically modified tumor-reactive TIL with one or more T-cell stimulating agent to produce a population of genetically modified and expanded tumor-reactive TIL; and (e) harvesting the population of genetically modified and expanded tumor-reactive TIL from step (d). In some embodiments, the one or more T cell activation markers are PD-1 and / or CD39, such as both PD-1 and CD39.

[0286] The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead and / or are detectably labeled, specifically bind to cell surface molecules if present on cells within the sample. In some aspects, cells bound to the antibodies can be recovered or separated from non-bound cells in the sample.

[0287] In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps. Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[0288] In some embodiments, the T cells for use in connection with the provided methods can be enriched or sorted a variety of ways including, but not limited to, magnetic bead separation, fluorescent cell sorting, and disposable closed cartridge based cell sorters. In particular aspects, one or more reagents specific to T cells or a subset thereof, such as reagents specific to T cell activation markers for selecting reactive cells, can be used including, but not limited to, florescent antibodies, nanoparticles or beads on cell selection equipment, but not limited to, the CliniMACS, Sony FX500 or the Tyto cell sorting systems (Miltenyi).

[0289] In particular embodiments, selection of cells is carried out by flow cytometry-based cell sorting. Compared to other methods, flow cytometry-based cell sorting has the advantage that cells can be isolated in a single step on the basis of multiple parameters for each cell, thereby achieving a higher yield of cells and a higher purity that may not be possible with beadbased (e.g. magnetic bead-based) separations. Further, multiparameter cell staining and separation allows simultaneous labeling and identification and sorting of a plurality of antigens and characteristic fluorescent signals. Using flow cytometry sorting, a single process can remove and isolate specific populations based on a complex cell surface phenotype. Cell selection sorting equipment can be used that has a sufficiently high-throughput to handle large volumes and cell numbers. Non-limiting cell sorting equipment includes, for example, Sony FX500 orthe Tyto cell sorting systems (Miltenyi). For use in provided methods, the flow cytometer instrument is GMP compliant. Method of cell sorting to achieve multiparameter sorting for two or more cell surface markers (e.g. CD39 and PD-1) can be carried out using multicolor fluorophore reagents that are compatible. It is within the level of a skilled artisan to choose appropriate fluorophores and reagents, such as by choosing a bright fluorophore and choosing fluorophores that have minimal to no spectral overlap.

[0290] In certain embodiments, the sample is contacted with a binding agent, e.g., a detectably labeled binding agent, that specifically binds to a cell surface marker. In certain embodiments, the detectably labeled binding agent(s) are fluorescently labeled. In certain embodiments, T cells labeled with binding agents specific to a cell surface marker are identified by flow cytometry. In certain embodiments, the method further includes separating any resultant T cells labeled with the binding agent(s) from other components of the sample to produce a composition enriched for T cells surface positive for the one or more cell surface marker. Cell selection sorting equipment can be used that has a sufficiently high-throughput to handle large volumes and cell numbers. Non-limiting cell sorting equipment includes, for example, Sony FX500 or the Tyto cell sorting systems (Miltenyi).

[0291] In some embodiments, any antibody reagent used to select cells in accord with the provided methods is a GMP antibody reagent. In some embodiments, the reagent is an analyte specific reagent (ASR).

[0292] In some embodiments, cells are selected that are surface positive for CD39 and PD-1. In some cases, staining methods also can include selecting CD45, CD4 and / or CD8 T cells from a sample. In some embodiments, multiparameter flow cytometry is carried out. In some embodiments, a multiparameter flow cytometry involves a sequential gating strategy. In some cases, CD45 expression, which optionally can be coupled with side scatter, can be used to exclude CD45 negative cells such as red blood cells. In some embodiments, only lymphocytes are gated based on CD45 expression and their scattering (e.g. FSClow, SSClow). In some embodiments, T cells can be gated based on positive expression of CD4+ and CD8+ cells. Within the T cell population, cells positive for CD39 and PD-1 can be identified for sorting.

[0293] Methods and antibody reagents to select for cells positive for these markers are known and commercially available. Any of a variety of fluorophores can be coupled to the antibodies and used in the multiparameter flow cytometry. In some embodiments, multi-color staining or labeling is carried out using multiple fluorophores in which multiple stainingreagents against different cell surface markers are incubated with cells. In some embodiments, the fluorescent marker, e.g., conjugated to the one or more staining reagents, such as antibodies, are selected to minimize energy transfer between them, such as to avoid or minimize overlapping emission and absorption spectra. In some embodiments, each fluorescent marker has a different emission spectra. In some embodiments, the multiple fluorescent marker may be excited with a single wavelength or multiple wavelengths, but detection occurs in regions where the peak emission spectra do not overlap. In some embodiments, excitation of one or more of the fluorescent markers may be by light at a single or the same wavelength, but whereby different wavelengths of light are emitted therefrom.

[0294] In some embodiments, any fluorescent marker or fluorophore suitable for use with flow cytometry analysis can be used. Some non-limiting examples of fluorescent markers include fluorescent proteins (e.g., GFP, YFP, RFP), fluorescent moieties (e.g., fluorescein isothiocyanate) (FITC), Phycoerythrin (PE), allophycocyanin (APC), Alexa Fluor (AF)), nucleic acid colorants (e.g., 4 ', 6-diamidino-2-phenylindole (DAPI), SYT016, propidium iodide (PI), cell membrane stain (e.g., FMI-43), cell functional dyes (e.g., Fluo-4, Indo-1), and synthetic dyes (e.g., Brilliant Violet (BV)). Exemplary fluorophores include, but are not limited to, hydroxycoumarin, Cascade Blue, Dylight 405 Pacific Orange, Alexa Fluor 430, Fluorescein, Oregon Green, Alexa Fluor 488, BODIPY 493, 2,7-Diochlorofluorescien, ATTO 488, Chromeo 488, Dylight 488, HiLyte 488, Alexa Fluor 532, Alexa Fluor 555, ATTO 550, BODIPY TMR- X, CF 555, Chromeo 546, Cy3, TMR, TRITC, Dy547, Dy548, Dy549, HiLyte 555, Dylight 550, BODIPY 564, Alexa Fluor 568, Alexa Fluor 594, Rhodamine, Texas Red, Alexa Fluor 610, Alexa Fluor 633, Dylight 633, Alexa Fluor 647, APC, ATTO 655, CF633, CF640R, Chromeo642, Cy5, Dylight 650, Alexa Fluor 680, IRDye 680, Alexa Fluor 700 (AF700), Cy5.5, ICG, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7, PE-Cy7, Cy7.5, Alexa Fluor 790, Dylight 800, IRDye 800, BV421, BV510, BV570, BV605, BV650, BV711, BV750, BV785, Qdot® 525, Qdot® 565, Qdot® 605, Qdot® 655, Qdot® 705, or Qdot® 800. In some embodiments, the one or more fluorescent markers each individually comprise a fluorophore selected from the group consisting of PE-Cy7, APC, AF700, BV421, Aqua, and BV605.

[0295] Table 2 lists exemplary antibodies for use in staining and selection or sorting of cells as described herein.

[0296] In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and / or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence- activated cell sorting (FACS), including preparative scale (FACS) and / or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers (e.g., with a antibody-coupled fluorescent peptide) are carried in a fluidic stream.

[0297] In some embodiments, the cell staining involves incubation with an antibody or binding partner that specifically binds to such markers as described, which in some embodiments is followed by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. In some aspects of such processes, a volume of cells is mixed with an amount of a desired staining reagent and incubated under conditions for staining of the cells. In some embodiments, the staining or labelling is carried out at a temperature between 0°C and 25 °C, such as at or about 4°C. In some embodiments, the staining or labelling is carried out for greater than 5 minutes, typically greater than 15 minutes. In some embodiments, the staining or labelling is carried out for between 15 minutes and 6 hours, such as between 30 minutes and 2 hours. In some embodiments, the staining or labelling is carried out for example, at or about 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, or any value between any of the foregoing. In some embodiments, the labeling with the one or more staining reagents is carried out simultaneously. In some embodiments, one or more wash steps are carried out prior to introducing the sample into the flow cytometer for analysis.

[0298] In some embodiments, the cell sample is prepared by suspending single cells at a density of 1 x 106to 5 x 107cells / ml in order to allow the cells to pass through the flow cytometer for reading. In some embodiments, the density of cells for sorting is 5 x 106cells / mL to 50 x 106cells / mL, such as at or about 20 x 106cells / mL. In some embodiments, thisconcentration of cells is called the fluid sheath. In some embodiments, the fluid sheath influences the rate of flow sorting, which typically progresses at around 2,000-20,000 cells (events) per second. The cell sample's) fluid sheath is typically made of a phosphate buffered saline solution, but other solutions are available as will be known and understood by those skilled in the art.

[0299] In some embodiments, the flow cytometry sorting rate In some embodiments, the flow cytometry sorting rate is from 2000 events / second to 10,000 event / second.. In some embodiments, the flow cytometry sorting rate is about 2000 events / second, about 3000 events / second, about 4,000 events / second, about 5000 events / second, about 6000 events / second, about 7000 events / second, about 8000 events / second, about 9000 events / second, about 10000 events / second, about 15000 events / second, or about 20000 events per second, or any value between any of the foregoing. In some embodiments, the flow cytometry sorting rate is about 6,000 events / second.

[0300] In some embodiments, the sample is introduced into a flow cytometer. The cell sample is typically narrowed down to a single stream through a fluidics system with the application of hydro pressure. This stream is then passed through the one or more beams of light scattering or fluorescence emission. Lasers typically serves as the light source in flow cytometers. The laser produces a single wavelength of light that once contacted with the cell sample produces scattered light in the forward direction as a measure of cell size, scattered light in the side direction as a measure of cell complexity, and fluorescent light, also emitted in the side direction which is proportional to the relative amount of a particular cell marker. Fluorescent channels are usually indicated by the designations FL1, FL2, FL3, etc., depending on the number of channels in the instrument. Each fluorescent channel is set with barrier filters to detect a selected specific dye while filtering out all others. The channel in which a specific dye is predominantly detectable may be referred to as its primary fluorescent channel while other fluorescent channels may be designated as secondary channels. Scattered and fluorescent emitted light signals are converted to electronic pulses that are processed by the flow cytometry engine and displayed on a graphical user interface “GUI.”

[0301] Methods of analyzing flowcytometric or FACS data can involve a “gating” for data to separate specific groups of cells. Different cell types can be identified by the scatter parameters and the fluorescence emissions resulting from labeling various cell proteins with dye-labeled antibodies as described above. The identification of clusters and, thereby,populations can be carried out by “gating” of the cells. In some embodiments, gates corresponding to subsets of particles of interest, such as TIL expressing markers of neoantigen reactivity, are defined by users with the aid of software operationally associated with the flow system as described above.

[0302] In some embodiments, a gate may be a “threshold” gate, which is a gate for only one optical parameter that defines an open region within the multidimensional space. In some embodiments, “threshold” gating can been used for forward light scatter to remove high frequency low level signals caused by interference, such as debris in the sample. In some embodiments, “window” gating is employed, e.g., by defining upper and lower bounds for signal values. In some embodiments, gating is carried out on a 2D-plot of two parameters, such as side scatter (e.g., on vertical axis) and a fluorescence signal (e.g., on horizontal axis).

[0303] In some embodiments, flow cytometry for a cell surface marker includes gating for an “F-minus one” (FMO) control. FMO gating includes separate portions of the same sample stained with a panel of detectably labeled binding agents that contains all the agents but one. The distribution of the signal of the removed fluorophore can be used to define the positive threshold for the missing label as it is known that all cells are negative in the control. The position of all gates can be determined using FMO controls in which the antibody against the investigated marker is substituted with an appropriate isotype control. In an exemplary method, a gate can be drawn using cells stained with the FMO cocktail around cells positive for CD45, CD4 and / or CD8, PD1 and CD39. In some embodiments, sequential gates can be used to arrive at the selected subpopulation. For example, methods can be carried out in which cells are gated on CD45 positive cells, then CD4 and / or CD8 positive cells, then on cells positive for PD-1 and CD39. In some embodiments, a viability dye also can be added. An exemplary viability dye is 7-ADD. In some embodiments, a gate can be drawn around cells negative for 7-AAD (7- AADneg).

[0304] In some embodiments, the cells are sorted into a single population of cells and collected. The selected population of cells are used as input for expansion, such as described in Section II.C.

[0305] The selection need not result in 100 % enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasingthe number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker.

[0306] In some embodiments, the enriched population of cells are enriched cells from a starting sample as describe above, in which the percentage of cells of a particular phenotype, e.g. tumor-reactive CD3+ T cells or CD3+ T cells surface positive for one or more T cell marker, e.g. PD-1 andCD39, in the enriched population of cells in increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 500%, 1000%, 5000% or more greater than the percentage of such cells in the starting sample. In some embodiments, the purity of tumor- reactive CD3+ T cells or CD3+ T cells surface positive for PD-1 andCD39) versus total cells in the population of enriched cells, is at least 90%, 91%, 92%, 93%, 94%, and is generally at least 95%, 96%, 97%, 98%, 99% or greater.C. Stimulation or activation of T cells for Expansion (e.g. First Expansion)

[0307] In some embodiments, the stimulation of T cells for expansion before gene editing can be carried out using any of the methods employed in section I-E. In some embodiments, a stimulation of T cells for expansion is performed before genetic modification is performed on the selected cells. In some embodiments, selected cells are genetically modified without having gone through stimulation for T cell expansion. In some embodiments, the selected cells are not stimulated or expanded before being genetically modified. In some embodiments, the target site or gene which is being modified determines whether the selected cells are stimulated for expansion before and after the genetic modification or if they are only stimulated for expansion after the genetic modification.

[0308] The provided methods further include expanding or activating the population of selected TILs by culture with one or more T-cell stimulating agent of lymphocytes under conditions to produce a population of expanded T cells. In some embodiments, the T cell stimulating agents are T cell activating agents. Hence, the provided methods involve the ex vivo expansion and production of a T cell therapeutic composition, particularly for use in connection with treating cancer. In some embodiments, the provided methods include a first and a second expansion of the population of selected TILs by culture with one or more T-cell simulating agents of lymphocytes under conditions to produce a population of expanded T cells. In some embodiments, the first expansion happens before the TILs are genetically modified and the second expansion happens after the TILs are genetically modified. In some embodiments, theTILs are genetically modified without an expansion, then after they TILs are genetically modified, the TILs go through a single expansion.

[0309] In some embodiments, the incubation or culturing with one or more T cell stimulatory agent(s) results in expansion or outgrowth of selected T cells, or a desired subset or subtype thereof or for viable cells thereof, for use in subsequent steps of the provided methods. Non-limiting examples of T cell stimulatory agent(s) and conditions for incubation or culture are described herein and in Section I-E.

[0310] Thus, among the provided methods are methods of culturing T cells for manufacture of tumor reactive T cells in which T cells are cultured or incubated in the presence of a T cell stimulatory agent under conditions to expand T cells.

[0311] In some embodiments, the T cell stimulatory agent(s) include a recombinant T cell stimulating cytokine, such as IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27 and / or IL-35. In some embodiments, the T cell stimulating cytokine includes IL-2, alone or in combination with another cytokine from among IL-7, IL- 15, IL-21, IL-25, IL-23, IL- 27 and / or IL-35. In some embodiments, the T cell stimulatory agent(s) include a recombinant T cell stimulating cytokine, such as IL-2, IL-7, IL-15, IL-21, IL-25 and / or IL-23. In some embodiments, the T cell stimulating cytokine includes IL-2, alone or in combination with another cytokine from among IL-7, IL- 15, IL-21, IL-25 and / or IL-23. In some embodiments, the T cell stimulating cytokine is one, two, three or more of IL-2, IL-7, IL- 15 and IL-21. In some embodiments, the T cell stimulating cytokine includes IL-2, alone or in combination with another cytokine from among IL-7, IL- 15, and / or IL-21. In some embodiments, the T cell stimulating cytokine includes IL-2, alone or in combination with another cytokine from IL-25, IL-23, IL-27 and / or IL-35. In some embodiments, the T cell stimulating cytokines are IL-7 and IL- 15.

[0312] In some embodiments, the choice of cytokine or combination of cytokines is within the level of a skilled artisan, so long as the cytokines or cytokines provide activity to stimulate the T cells to expand. The activity to stimulate tumor reactive T cells can be direct or indirect. In some embodiments, the one or more cytokines directly stimulate tumor reactive T cells to expand or proliferate. In some embodiments, the one or more cytokines suppress T regulatory T cells, thereby indirectly stimulating or enhancing proliferation of desired tumor reactive T cells. In some embodiments, the T-cell stimulating agents include anti-CD3 (e.g. anti-CD3 antibody, such as OKT3), anti-CD28 reagents (e.g. anti-CD28 antibody), such as an anti-CD3 antibody (e.g. OKT3) and an anti-CD28 antibody and / or one or more recombinant cytokine (e.g. IL-2, IL-7, IL-21 and / or IL- 15) . In some embodiments, the T-cell simulating agents include anti-CD3 antibody but not an anti-CD28 antibody.

[0313] In some of any of the provided embodiments, the T cell stimulatory agent(s) is selected from an agent that initiates TCR / CD3 intracellular signaling and an agent that initiates signaling via a costimulatory receptor. In some of any of the provided embodiments, the agent that initiates TCR / CD3 intracellular signaling is an anti-CD3 antibody, such as OKT3. In some of any of the provided embodiments, the agent that initiates signaling via a costimulatory receptor comprises peripheral blood mononuclear cells (PBMCs), optionally non-dividing or irradiated PBMCs. In some of any of the provided embodiments, the agent that initiates signaling via a costimulatory receptor is an anti-CD28 antibody. In some of any of the provided embodiments, the T cell stimulatory agent(s) is an anti-CD3 antibody and an anti-CD28 antibody that each are soluble. In particular embodiments, one or more recombinant cytokines also are present as additional T cell stimulatory agents during the incubation. In some embodiments, the incubation with a T cell stimulatory agent(s) include incubation with at least one T cell stimulating recombinant cytokine (e.g. recombinant IL-2, IL-7, IL-21, IL-15, IL-25, IL-23, IL- 27, and / or IL-35) and a further T cell stimulatory agent(s) that engage CD3 and / or a costimulatory molecule (e.g. CD28) on T cells.

[0314] In embodiments of the provided methods, the stimulating conditions include one or more agent, e.g., ligand, which turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell and / or a costimulatory signal in a T cell. Such agents can include antibodies, such as those specific for a TCR component, e.g., anti-CD3, and / or costimulatory receptor, e.g. anti- CD28 or anti-4- IBB. In some embodiments, such agents are added to the culture medium as soluble antibodies. In other embodiments, such agents are bound to solid support such as a bead. In some embodiments, the T cell stimulatory agent(s) includes anti-CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander). In some embodiments, the T cell stimulatory agent(s) includes anti-CD3 beads and anti-4- IBB beads .

[0315] An anti-CD3 antibody can include any antibody directed against or that can specifically bind the CD3 receptor on the surface of T cells, typically human CD3 on human T cells. Anti-CD3 antibodies include OKT3, also known as muromonab. Anti-CD3 antibodies also include the UHCTI clone, also known as T3 and CD3E. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab. The anti-CD3 antibody can be addedas a soluble reagent or bound to a bead. In particular embodiments, the anti-CD3 antibody is soluble.

[0316] In some embodiments, the T cell stimulatory agent(s) includes incubation with an anti-CD3 antibody and incubation with a further agent that specifically binds to CD28 or stimulates or induces a CD28-mediated signal in cells. In some embodiments, the CD28- mediated signal can be initiated or provided by anti-CD28 antibody or antigen-binding fragment thereof. In some embodiments, the CD28-mediated signal can be provided by antigen- presenting feeder cells (APCs), such as peripheral blood mononuclear cells (PBMC).

[0317] In some embodiments, the T cell stimulatory agent(s) includes incubation with an anti-CD3 antibody and incubation with a further agent that specifically binds to 4- IBB or stimulates or induces a 4- IBB -mediated signal in cells. In some embodiments, the 4-1BB- mediated signal can be initiated or provided by anti-4- IBB antibody or antigen-binding fragment thereof. In some embodiments, the 4-lBB-mediated signal can replace antigen- presenting feeder cells (APCs), such as peripheral blood mononuclear cells (PBMC). In some embodiments, the 4- IBB antibody or antigen-binding fragment thereof is an agonist of 4- IBB. In some embodiments, the 4-1BB antibody may be Urelumab or BMS-663513.

[0318] In some embodiments, the T cell stimulatory agent(s) can include adding to the population of T cells feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC). In some aspects, the non-dividing feeder cells can comprise gamma- irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells. In some embodiments, the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded. In some embodiments, the ratio of T cells to PBMCs and / or antigen-presenting cells is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500.

[0319] In some embodiments, the stimulation does not include incubation with PBMCs or other feeder cells, such as non-divided or irradiated PBMCs or other non-dividing or irradiated feeder cells.

[0320] In some embodiments, the T cell stimulatory agent(s) include one or more recombinant cytokine. In some embodiments, the cytokine is added or is exogenous to the culture media. Thus, in some embodiments, one or more further recombinant cytokine also is included during the culturing. In some embodiments, the recombinant cytokine can include one or more of IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27 and / or IL-35. In some embodiments, the recombinant cytokine can include one or more of IL-2, IL-7, IL- 15, IL-21, IL-25 and / or IL- 23. In some embodiments, the culturing and incubation is carried out in the presence of recombinant IL-2, IL- 15 and IL-7. In some embodiments, the culturing is carried out in the presence of a IL-2. In some embodiments, the culturing is carried out in the presence of IL- 15 and IL- 17, which, in some aspects does not additionally include IL-2. In particular embodiments, the recombinant cytokine(s) is human.

[0321] The recombinant cytokine generally is a recombinant human protein. In particular embodiments, the recombinant cytokine is present in the cell culture medium during the incubation at a concentration of at least or at least about 0.5 lU / mL, at least or at least about 1.0 lU / mL, at least or at least about 5 lU / mL, at least at or about or at or about 10 lU / mL, at least at or about or at or about 100 lU / mL, at least at or about or at or about 1000 lU / mL, at least at or about or at or about 1500 lU / mL, at least at or about or at or about 2000 lU / mL, at least at or about or at or about 2500 lU / mL, at least at or about or at or about 3000 lU / mL, at least at or about or at or about 3500 lU / mL, at least at or about or at or about 4000 lU / mL, at least at or about or at or about 4500 lU / mL, at least at or about or at or about 5000 lU / mL, at least at or about or at or about 5500 lU / mL, at least at or about or at or about 6000 lU / mL, at least at or about or at or about 6500 lU / mL, at least at or about or at or about 7000 lU / mL, at least at or about or at or about 7500 lU / mL, or at least at or about or at or about 8000 lU / mL. In an embodiment, the cell culture medium comprises between at or about 10 lU / mL and at or about 100 lU / mL, at or about 100 lU / mL and at or about 1000 lU / mL, at or about 1000 and at or about 2000 lU / mL, between at or about 2000 and at or about 3000 lU / mL, between at or about 3000 and 4000 at or about lU / mL, between at or about 4000 and at or about 5000 lU / mL, between at or about 5000 and at or about 6000 lU / mL, between at or about 6000 and at or about 7000 lU / mL, between at or about 7000 and at or about 8000 lU / mL, each inclusive.

[0322] In some embodiments, recombinant IL-2 is present in the cell culture medium. In some aspects, IL-2 is the only recombinant cytokine added to the culture. In some aspects, recombinant IL-2 and one other recombinant modulatory cytokine from IL-7, IL- 15, IL-21, IL-23, IL-25, IL- 27 or IL-35 is added to the culture. IL-2 is a cytokine that supports T cell recovery and proliferation. IL-2 also supports the homeostasis of T cells, thereby supporting their phenotype, differentiation status, and immune memory. In some cases, induction of regulatory T cells in the tumor microenvironment may lead to low bioavailability of IL-2. Recombinant IL-2 has been regularly used in broad expansion of T cells in various contexts. Recombinant IL-2 is commercially available. In particular embodiments, recombinant IL-2 is GMP grade (e.g. MACS GMP Recombinant Human IL-2, Miltenyi Biotec).

[0323] Recombinant IL-2 can be included in cell culture media during various stages of the provided process. In some cases, recombinant IL-2 can be included in the initial T cell expansion (first expansion), such as to promote TIL outgrowth

[0324] In some embodiments, recombinant IL- 15 is present in the cell culture medium. IL- 15 is a cytokine that is involved in memory T cell homeostasis and activation. In some cases, IL- 15 can promote effector functions of antigen-experienced T cells in the absence of antigen and prevent their differentiation into an exhausted phenotype. IL- 15 also plays a role in T cell proliferation. Recombinant IL- 15 is commercially available. In particular embodiments, recombinant IL-15 is GMP grade (e.g. MACS GMP Recombinant Human IL-15, Miltenyi Biotec).

[0325] Recombinant IL- 15 can be included in cell culture media during various stages of the provided process. In some cases, recombinant IL-15 can also be included in cultures to expand tumor-reactive T cells during the expansion phase. In some cases, recombinant IL- 15 can be combined with recombinant IL-7 to provide for activation, survival and / or expansion of tumor- reactive T cells in the provided methods. In some such embodiments, the combination of recombinant IL-7 and IL- 15 is an alternative to the use of recombinant IL-2 in the culture, and the culture media does not additionally contain recombinant IL-2.

[0326] In some embodiments, the initial expansion (e.g. first expansion) is carried out in the presence of recombinant IL- 15 added at a concentration of 500 lU / mL to 2000 lU / mL (e.g. at or about 1000 lU / mL). In some embodiments, the initial expansion (e.g. first expansion) is carried out in the presence of recombinant IL-15 added at a concentration of at or about 1000 lU / mL. In some embodiments, at least one other recombinant modulatory cytokine from IL-2, IL-7, IL-21, IL-23, IL-25, IL- 27 or IL-35 is added to the culture medium.

[0327] In some embodiments, recombinant IL-7 is added to the culture medium. In some aspects, recombinant IL-7 is added to the culture media with one or both of IL-2 or IL- 15. Insome aspects, recombinant IL-7 and recombinant IL-2 are added to the culture media. In some aspects, recombinant IL-7 and recombinant IL- 15 are added to the culture media. In some aspects, recombinant IL-7 (e.g. in combination with one or both of IL-2 and IL-15) and one other recombinant modulatory cytokine from IL-23, IL-25, IL- 27 or IL-35 is added to the culture medium. IL-7 is a cytokine that is involved in promoting T cell maintenance and homeostasis. In some cases, IL-7 can boost memory T cell survival and proliferation, particularly the central memory compartment. Recombinant IL-7 is commercially available. In particular embodiments, recombinant IL-7 is GMP grade (e.g. MACS GMP Recombinant Human IL-7, Miltenyi Biotec).

[0328] Recombinant IL-7 can be included in cell culture media during various stages of the provided process. In some cases, recombinant IL-7 can also be included in cultures to expand tumor-reactive T cells during the expansion phase. Inclusion of recombinant IL-7 in the process can maintain or support expansion of memory T cell subsets in the process. In some cases, recombinant IL-7 can be combined with recombinant IL- 15 to provide for activation, survival and / or expansion of tumor-reactive T cells in the provided methods. In some such embodiments, the combination of recombinant IL-7 and IL- 15 is an alternative to the use of recombinant IL-2 in the culture, and the culture media does not additionally contain recombinant IL-2.

[0329] In some embodiments, recombinant IL- 15 and IL-7 are added to the culture medium. In some embodiments, recombinant IL- 15 is added at a concentration of 500 lU / mL to 2000 lU / mL (e.g. at or about 1000 lU / mL) and recombinant IL-7 is added at a concentration of 400 lU / mL to 2000 lU / mL (e.g. at or about 600 lU / mL or 1000 lU / mL). In some embodiments, the initial expansion (e.g. first expansion) is carried out in the presence of recombinant IL- 15 added at 1000 lU / mL and recombinant IL-7 added at 1000 lU / mL. In some embodiments, the first expansion is carried out in the presence of recombinant IL- 15 added at 1000 lU / mL and recombinant IL-7 added at 600 lU / mL. In some embodiments, at least one other recombinant modulatory cytokine from IL-2, IL-21, IL-23, IL-25, IL-27 or IL-35 is added to the culture medium.

[0330] In some embodiments, recombinant IL-21 is added to the culture medium. In some aspects, recombinant IL-21 is added to the culture media with one or both of IL-2, IL-7, or IL- 15. In some aspects, recombinant IL-21 and recombinant IL-2 are added to the culture media. In some aspects, recombinant IL-21 and recombinant IL- 15 are added to the culture media. In some aspects, recombinant IL-21 (e.g. in combination with one or more IL-2, IL-7 and IL- 15)and one other recombinant modulatory cytokine from IL-23, IL-25, IL-27 or IL-35 is added to the culture medium. IL-21 is a cytokine that supports a broad range of T cell activation without increasing regulatory T cell signaling. In some cases, IL-21 can support memory cell stabilization, effector function, and proliferation of antigen-experienced T cells. IL-21 can induce upregulation of effector molecules in both CD4 and CD8 T cells. Recombinant IL-21 is commercially available. In particular embodiments, recombinant IL-21 is GMP grade (e.g. MACS GMP Recombinant Human IL-21, Miltenyi Biotec).

[0331] Recombinant IL-21 can be included in cell culture media during various stages of the provided process. In some cases, recombinant IL-21 can also be included in cultures to expand tumor-reactive T cells during the expansion phase, such as to support proliferation and stabilization of memory phenotype. In some such embodiments, the combination of recombinant IL-21 and IL- 15 is an alternative to the use of recombinant IL-2 in the culture, and the culture media does not additionally contain recombinant IL-2.

[0332] In one aspect of provided methods, the tumor reactive T cells are directly sorted after tumor digest before being genetically modified, and only a single expansion step is carried out after the genetic modification, in which the population of expanded T cells is harvested as a therapeutic TIL composition. In such an example, tumor fragments are digested into a single cell suspension and provided as an input sample for sorting / selection for the tumor-reactive T cells thereof. Then, the selected cells are genetically modified using any of the techniques described in Section LD before the cells are expanded using any of the techniques here or in section LE and harvested as a therapeutic TIL composition. In some embodiments, the expansion is carried out for a period of time to achieve a therapeutic dose. In some embodiments, the expansion is carried to achieve a fold expansion of the cells of from at or about 200-fold to at or about 3000- fold. In some embodiments, the expansion is carried out to achieve a therapeutic dose of at or about or greater than at or about 500 million total cells. In some embodiments, the activation and / or expansion is carried out for 1-28 days, such as for at or about 7 to 28 days, 7 to 21 days, 7 to 14 day, such as at or about 7 days, 8 days, 9 days, 10 days, 11 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days or 28 days. In some embodiments, the activation and / or expansion is carried out for 1 to 21 days. In some embodiments, the activation and / or expansion is carried out for 1 to 6 days. In some embodiments, the activation and / or expansion is carried out for 4 to 10 days.In some embodiments, the activation and / or expansion is carried out for 8 to 14 days. In some embodiments, the activation and / or expansion is carried out for 11 to 17 days.

[0333] In some embodiments, provided herein is a method for manufacturing tumor-reactive T cells comprising (a) selecting cells surface positive for PD-1 and / or CD39 from an input sample comprising T cells from a subject that has a tumor to obtain selected cells from the sample; (b) performing an expansion by culture of the selected cells with one or more T-cell stimulating agent of lymphocytes under conditions to produce a population of expanded T cells; (c) genetically modifying the expanded cells; and (d) performing another expansion by culture of the genetically modified cells with one or more T-cell simulating agent of lymphocytes under conditions to produce a population of expanded T cells. In some embodiments, provided herein is a method for manufacturing tumor-reactive T cells comprising (a) selecting cells surface positive for PD-1 and / or CD39 from an input sample comprising T cells from a subject that has a tumor to obtain selected cells from the sample; (b) genetically modifying the expanded cells; and (c) performing an expansion by culture of the genetically modified cells with one or more T- cell simulating agent of lymphocytes under conditions to produce a population of expanded T cells. In some embodiments, the method includes harvesting the population of expanded T cells produced by the method for formulation as the therapeutic composition.

[0334] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells, contains recombinant IL-2. In some embodiments, one or more other stimulating agent can be included such as one or more other recombinant cytokine from IL-7, IL-15, IL-21, IL-25, and / or IL-23, or an anti-CD3 antibody (e.g. OKT-3). In some cases in which an anti-CD3 antibody (e.g. OKT-3) the T cell stimulating agent(s) also can include a costimulating agent, such as provided by antigen-presenting feeder cells, such as PBMCs, or a soluble anti-CD28 antibody.

[0335] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL-2 and an anti-CD3 antibody.

[0336] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL-2, an anti-CD3 antibody, e.g. OKT-3, and antigen-presenting feeder cells, such as PBMCs.

[0337] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL- 15, IL-21 and an anti-CD3 antibody.

[0338] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL-15, IL-21, an anti-CD3 antibody, e.g. OKT-3, and antigen-presenting feeder cells, such as PBMCs.

[0339] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL- 15, IL-7 and an anti-CD3 antibody.

[0340] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL-15, IL-7, an anti-CD3 antibody, e.g. OKT-3, and antigen-presenting feeder cells, such as PBMCs.

[0341] In some embodiments, the stimulated cells are collected and are cryofrozen. In some embodiments, for cryopreservation, the stimulated cells are formulated as a composition with a cryoprotectant. In some embodiments, the cryoprotectant is or comprises DMSO and / or glycerol. In some embodiments, compositions formulated for cryopreservation can be stored at low temperatures, such as ultra low temperatures, for example, storage with temperature ranges from -40 °C to -150 °C, such as or about 80 °C ± 6.0 ° C.

[0342] In some embodiments, the cryopreserved cells are prepared for subsequent steps by thawing.D. Genetically Modifying TILs

[0343] In some embodiments, the gene editing is carried out on a population of tumor- reactive cells obtained from a patient’s tumor. In some embodiments, the cells to be genetically modified are directly obtained from a subjects tumor. In some embodiments, the gene editing is carried out on tumor reactive cells that have been enriched from the tumor by selection of cell surface positiove for one or more T cell activation marker(selected cells) such as cells selected by methods described in Section I.B. In some embodiments, the gene editing is carried out on a population of any of such cells that have been subjected to expansion such as in accord with methods in Section I.C.

[0344] In some embodiments, prior to introducing a genetic disruption at a target site of a target gene or loci , the population of tumor-reactive TIL are contacted with a T cell activation reagent for activation of cells in the population. In some embodiments, the T cell activation reagent comprises an anti-CD3 antibody or antigen binding fragment thereof. In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof is a primary activation agonist and the T cell activation reagent further comprises a costimulatory agonist. In some embodiments, the costimulatory agonist is an anti-CD28 antibody or antigen binding fragmentthereof. In some embodiments, the T cell activation reagent comprises an anti-CD3 antibody and an anti-CD28-antibody. In some embodiments, the anti-CD3 / anti-CD28 antibodies are immobilized on beads (e.g., Dynabeads). In some embodiment, the T cell activation reagent comprises a costimulatory agonist. In some embodiments, the costimulatory agonist is a 4- IBB agonist. In some embodiments, the costimulatory agonist is an anti-4- IBB antibody or antigen binding fragment thereof. In some embodiments, the T cell activation reagent comprises an anti- CD3 antibody and an anti-4- IBB antibody. In some embodiments, the T cell activation reagent comprises an anti-CD3 antibody or antigen binding fragments thereof and an anti-4- IBB antibody or antigen binding fragment thereof, that are immobilized on beads. In some embodiments, after the T cells are contacted with the T cell activation reagent they are allowed to rest for 2 to 6 days, as at or about 3 days prior to subjecting the cells to gene editing. In some embodiments, the population of tumor-reactive TIL are introduced with the genetic disruption within about 3 days of contacting with the T cell activation reagent.

[0345] In some embodiments, the contacting with a T cell activation reagent is carried out for 1-28 days, such as for at or about 7 to 28 days, 7 to 21 days, 7 to 14 day, such as at or about 1 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days or 28 days. In some embodiments, the contacting with a T cell activation reagent is carried out for 1 to 21 days. In some embodiments, the contacting with a T cell activation reagent is carried out for 1 to 6 days. In some embodiments, contacting with a T cell activation reagent is carried out for 4 to 10 days. In some embodiments, the contacting with a T cell activation reagent is carried out for 8 to 14 days. In some embodiments, the contacting with a T cell activation reagent is carried out for 11 to 17 days. In some embodiments, the contacting with a T cell activation reagent is carried out for 11 days. In some embodiments, the contacting with a T cell activation reagent is carried out for 7 days. In some embodiments, the contacting with a T cell activation reagent is carried out for 3 days.

[0346] In some embodiments, the contacting with a T cell activation reagent is a first contacting or first activation with a first T cell activation reagent and the method further comprises a second contacting or second activation with a second T cell activation reagent. In some embodiments, the second T cell activation reagent comprises an anti-CD3 antibody or antigen binding fragment thereof. In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof is a primary activation agonist and the second T cell activation reagentfurther comprises a costimulatory agonist. In some embodiments, the costimulatory agonist is an anti-CD28 antibody or antigen binding fragment thereof. In some embodiments, the second T cell activation reagent comprises an anti-CD3 antibody and an anti-CD28-antibody. In some embodiments, the anti-CD3 / anti-CD28 antibodies are immobilized on beads (e.g., Dynabeads). In some embodiment, the second T cell activation reagent comprises a costimulatory agonist. In some embodiments, the costimulatory agonist is a 4- IBB agonist. In some embodiments, the costimulatory agonist is an anti-4- IBB antibody or antigen binding fragment thereof. In some embodiments, the second T cell activation reagent comprises an anti-CD3 antibody and an anti- 4-1 BB antibody.

[0347] In some embodiments, the contacting with the second T cell activation reagent is carried out for 1-28 days. In some embodiments, the contacting with the second T cell activation is carried out for any of the times described above for contacting. In some embodiments, the first T cell activation reagent comprises an anti-CD3 antibody or antigen binding fragment thereof and an anti-CD28 antibody or antigen binding fragment thereof. In some embodiments, the second T cell activation reagent comprises an anti-CD3 antibody or antigen binding fragment thereof and an anti-4- IBB antibody or antigen binding fragment thereof.

[0348] In some embodiments, TILs are genetically modified by genetic disruption of a target site in a gene of interest, such as those described below, which leads to a mutation preventing transcription or translation of the edited gene or causes the protein product to not function, leading to a knockout of the gene of interest in the TILs. In some embodiments, the genetic disruption results in an insertion / deletion (indel) in the target gene. In some embodiments, the genetic disruption is an all alleles, such as both alleles, of the target gene. In some embodiments, the genetic disruption inactivates the target gene. In some embodiments, the genetic disruption results in knockout of expression of the target gene. In some embodiments, gene expression is knocked out or eliminated in the cell. In some embodiment, surface protein expression of the target gene is knocked out or eliminated in the cell. / . Target loci and guide UNAs

[0349] In some embodiments, the provided methods include introducing a genetic disruption at a target site within a target gene in a T cell selected from the group consisting of SOCSL GISH. PDCD1 (PD-1), TIGIT, RC3H1 (Roquin), CMIP (gene encoding C-Maf Inducing Protein), CD39, LAG3, TIM3, MAPK14, and RASA2 (gene encoding RAS p21 protein activator 2). In some embodiments, the target gene is SOCS1. In some embodiments, the target gene isCISH. In some embodiments, the target gene is PD-1. In some embodiments, the target gene is TIGIT. In some embodiments, the target gene is Roquin (R3CH1). In some embodiments, the target gene is CMIP. In some embodiments, the target gene is CD39. In some embodiments, the target gene is LAG3. In some embodiments, the target gene is TIM3. In some embodiments, the target gene is MAPK14. In some embodiments, the target gene is RASA2.

[0350] In some embodiments, the provided methods include introducing a genetic disruption at a target site within the gene S0CS1 in a T cell. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 13-22. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the gRNA comprises a spacer with a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 35-44. In some embodiments, the provided methods include introducing a genetic disruption at a target site with a gRNA, wherein the target site is selected from a sequence set forth in SEQ ID NOs: 13-22.

[0351] In some embodiments, the provided methods include introducing a genetic disruption at a target site within the gene CISH in a T cell. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 1-7. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the gRNA comprises a spacer with a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 23-29. In some embodiments, the provided methods include introducing a genetic disruption at a target site with a gRNA, wherein the target site is selected from a sequence set forth in SEQ ID NOs: 1-7.

[0352] In some embodiments, the provided methods include introducing a genetic disruption at a target site within the gene PD-1 in a T cell. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the target site is asequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 8-12. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the gRNA comprises a spacer with a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 30-34. In some embodiments, the provided methods include introducing a genetic disruption at a target site with a gRNA, wherein the target site is selected from a sequence set forth in SEQ ID NOs: 8-12.

[0353] In some embodiments, the provided methods include introducing a genetic disruption at a target site within the gene TIGIT in a T cell. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 59-63. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the gRNA comprises a spacer with a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 100-104. In some embodiments, the provided methods include introducing a genetic disruption at a target site with a gRNA, wherein the target site is selected from a sequence set forth in SEQ ID NOs: 59-63.

[0354] In some embodiments, the provided methods include introducing a genetic disruption at a target site within the gene RC3H1 in a T cell. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 79-83. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the gRNA comprises a spacer with a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 120-124. In some embodiments, the provided methods include introducing agenetic disruption at a target site with a gRNA, wherein the target site is selected from a sequence set forth in SEQ ID NOs: 79-83.

[0355] In some embodiments, the provided methods include introducing a genetic disruption at a target site within the gene CMIP in a T cell. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 89-94. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the gRNA comprises a spacer with a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 130-135. In some embodiments, the provided methods include introducing a genetic disruption at a target site with a gRNA, wherein the target site is selected from a sequence set forth in SEQ ID NOs: 89-94.

[0356] In some embodiments, the provided methods include introducing a genetic disruption at a target site within the gene CD39 in a T cell. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 84-88. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the gRNA comprises a spacer with a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 125-129. In some embodiments, the provided methods include introducing a genetic disruption at a target site with a gRNA, wherein the target site is selected from a sequence set forth in SEQ ID NOs: 84-88.

[0357] In some embodiments, the provided methods include introducing a genetic disruption at a target site within the gene LAG3 in a T cell. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 54-58. In some embodiments, the provided methods include introducing agenetic disruption at a target site wherein the gRNA comprises a spacer with a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 95-99. In some embodiments, the provided methods include introducing a genetic disruption at a target site with a gRNA, wherein the target site is selected from a sequence set forth in SEQ ID NOs: 54-58.

[0358] In some embodiments, the provided methods include introducing a genetic disruption at a target site within the gene TIM3 in a T cell. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 64-68. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the gRNA comprises a spacer with a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 105-109. In some embodiments, the provided methods include introducing a genetic disruption at a target site with a gRNA, wherein the target site is selected from a sequence set forth in SEQ ID NOs: 64-68.

[0359] In some embodiments, the provided methods include introducing a genetic disruption at a target site within the gene MAPK14 in a T cell. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 69-73. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the gRNA comprises a spacer with a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 110-114. In some embodiments, the provided methods include introducing a genetic disruption at a target site with a gRNA, wherein the target site is selected from a sequence set forth in SEQ ID NOs: 69-73.

[0360] In some embodiments, the provided methods include introducing a genetic disruption at a target site within the gene RASA2 in a T cell. In some embodiments, the provided methodsinclude introducing a genetic disruption at a target site wherein the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 74-78. In some embodiments, the provided methods include introducing a genetic disruption at a target site wherein the gRNA comprises a spacer with a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NOs: 115-119. In some embodiments, the provided methods include introducing a genetic disruption at a target site with a gRNA, wherein the target site is selected from a sequence set forth in SEQ ID NOs: 74-78.

[0361] In some embodiments, the target gene is a gene in which reduced expression of the gene leads to increased cytotoxic abilities of a genetically modified cell. In some embodiments, the target gene is a gene in which reduced expression of the gene leads to increased expansion of a genetically modified cell. In some embodiments, the target gene is a gene in which reduced expression of the gene leads to reduced reliance on one or more cytokines in media to expand a genetically modified cell.

[0362] In some aspects, the phenotype is assessed by killing assays, cell growth in media with or without one or more cytokines, apoptotic markers, or cell surface markers. In some embodiments, the target gene is knocked out. It is understood that a cell that has a gene knocked out is a cell were the gene has little to no expression of the target gene and little to no protein that would be transcribed from the target gene detected. Gene expression assays (e.g. qPCR, sequence validation, or RNAseq) along with protein quantification (e.g. Western Blot or mass spectrometry) could be used to assess if the target gene was knocked out

[0363] In some embodiments, the phenotype of genetically modifying a cell is increased cell expansion without one or more cytokines. In some embodiments, the phenotype of genetically modifying a cell is increased cell expansion in the absence of IL-2.

[0364] In some embodiments, the genetic modification is a genetic disruption. In some embodiments, the genetic modification is carried out with a CRISPR-Cas system or a DNA- targeting system that utilizes a guide RNA, which will bind to a target site to direct the CRISPR- Cas system or DNA-targeting system to specifically modify the genomic location at or near the target site. In some embodiments, the genetic modification or genetic disruption is carried out with a CRISPER-Cas system such as any described in Section LD-3.

[0365] In some embodiments, the genetic modification or genetic disruption is carried out in a gene expressed in response to T cell receptor or cytokine receptor signaling. In some embodiments, the genetic modification or genetic disruption is carried out in a gene that mediates JAK / STAT degradation. In some embodiments, the genetic modification or genetic disruption leads to superior control of tumor growth. In some embodiments, the genetic modification or genetic disruption leads to increased cytokine secretion. In some embodiments, the genetic modification or genetic disruption leads to increased responsiveness to IL- 12. In some embodiments, the genetic modification or genetic disruption is carried out in SOCS1. In some embodiments, the genetic modification or genetic disruption is within the KIR domain of the SOCS1 gene. In some embodiments, the KIR domain is involved in targeting the substratebinding groove of JAK. In some embodiments, the genetic modification or genetic disruption is within six continuous residues of the KIR domain of SOCS1 which binds to the substratebinding groove of JAK.

[0366] In some embodiments the target site is on chromosome 16. In some embodiments the target site is on chromosome 16 at p 13.13. In some embodiments, the target site is within SOCS1. In some embodiments, the target site is within exon 2 of SOCS1. In some embodiments, the target site is within a portion of the SOCS1 gene, which when translated constitutes a portion within the SH2 domain of the SOCS1 protein. In some embodiments, the target site is within a portion of the SOCS1 gene, which when translated constitutes a portion within the KIR domain of the SOCS1 protein. In some embodiments, the target site is one of the target sites listed in Table 3. In some embodiments, the gRNA that targets the target site comprises the SEQ ID NO of any that are listed in Table 3. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chrl6:ll, 255, 016-11, 255, 035. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chrl6:l l, 255, 013-11, 255, 032. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chrl6: 11,255,236-11,255,255. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chrl6:l l, 255, 230-11, 255, 249. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp ofgenomic coordinates chrl6:255, 037-11,255,056. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chrl6:l l, 255, 310-11, 255, 329. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chrl6:l l, 255, 292-11, 255, 311. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chrl6:l l, 255, 287-11, 255, 306. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chrl6:l l, 255, 117-11, 255, 139. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chrl6:l l, 255, 050-11, 255, 072.

[0367] In some embodiments, the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence selected from SEQ ID NOs: 13- 22. In some embodiments, the spacer of the gRNA is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NO: 35-44. In some embodiments, the spacer of the gRNA is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence as set forth in SEQ ID NO: 40. In some embodiments, the spacer of the gRNA is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence as set forth in SEQ ID NO: 41. In some embodiments, the spacer of the gRNA is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence as set forth in SEQ ID NO: 42. In some embodiments, the spacer of the gRNA is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence as set forth in SEQ ID NO: 43. In some embodiments, the spacer of the gRNA is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence as set forth inSEQ ID NO: 44. In some embodiments, the spacer of the gRNA is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence as set forth in SEQ ID NO: 35. In some embodiments, the spacer of the gRNA is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence as set forth in SEQ ID NO: 36. In some embodiments, the spacer of the gRNA is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence as set forth in SEQ ID NO: 37.

[0368] In some embodiments, the target site is adjacent to a protospacer adjacent motif (PAM). In some embodiments, the PAM sequence is or comprises “NGG” or is or comprises SEQ ID NO: 47. In some embodiments, the PAM sequence is or comprises “NNGRRT” or is or comprises SEQ ID NO: 48. In some embodiments, the PAM sequence is or comprises “TTTN”. In some embodiments, the PAM sequence is or comprises “YTTN”. In some embodiments, the PAM sequence is or comprises “YTTV” or is or comprises SEQ ID NO: 53. In some embodiments, the gRNA comprises a scaffold for binding to a Cas protein with a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of the sequence set forth in SEQ ID NO: 45.Table 3

[0369] In some embodiments, the target site is within a gene that is induced by T cell receptor stimulation. In some embodiments, the target site is within a gene that encodes a protein that interacts with PLC-yl. In some embodiments, the target site is within a gene that encodes a protein that causes degradation of the T cell receptor signaling intermediate. In some embodiments, the target site is within a gene that encodes a protein which reduces signaling from the T cell receptor. In some embodiments, the target site is within a gene that when expression or protein levels are reduced or deleted leads to enhanced CD8+T cell expansion. In some embodiments, the target site is within a gene that when expression or protein levels are reduced or deleted leads to enhanced functional avidity of the cell. In some embodiments, the target site is within a gene that when expression or protein levels are reduced or deleted leads to enhanced cytokine poly functionality. In some embodiments, the target site is within a gene that when expression or protein levels are reduced or deleted in sorted cells leads to enhanced regression of tumors when the modified sorted cells are administered to a patient with the tumor compared to non-modified sorted cells.

[0370] In some embodiments, the target site is on chromosome 3. In some embodiments, the target site is on chromosome 3 at p21.2. In some embodiments, the target site is within CISH. In some embodiments, the target site is within exon 3 of CISH. In some embodiments, the target site is one of the target sites listed in Table 4. In some embodiments, the gRNA that targets the target site comprises the SEQ ID NO of any that are listed in Table 4. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chr3:50, 607, 985-50, 608, 004. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chr3:50,607,687- 50,607,706. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chr3:50, 607, 907-50, 607, 925. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chr3:50, 607, 907-50, 607, 925. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chr3:50, 607, 809-50, 607, 828. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chr3:50, 607, 681-50,607,700. Insome embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinatesChr3:50, 607, 888-50, 607, 907. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chr3:50,608, 113-50,608, 132.

[0371] In some embodiments, the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence selected from SEQ ID NOs: 1-7. In some embodiments, the spacer of the gRNA is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NO: 23-29. In some embodiments, the spacer of the gRNA is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence as set forth in SEQ ID NO: 27.

[0372] In some embodiments, the target site is adjacent to a protospacer adjacent motif (PAM). In some embodiments, the PAM sequence is or comprises “NGG” or is or comprises SEQ ID NO: 47. In some embodiments, the PAM sequence is or comprises “NNGRRT” or is or comprises SEQ ID NO: 48. In some embodiments, the gRNA comprises a scaffold for binding to a Cas protein with a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of the sequence set forth in SEQ ID NO: 45. In some embodiments, the gRNA comprises a scaffold for binding to a Cas protein with a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of the sequence set forth in SEQ ID NO: 46.Table 4

[0373] In some embodiments, decreasing expression or protein levels of PD-1 in sorted cells decreases tumor burden are administered. In some embodiments, knocking out PD-1 in sorted cells decreases tumor burden after cells are administered. In some embodiments, decreasing expression or protein levels of PD-1 in sorted cells enhances the sorted cells anti-tumor immunity.

[0374] In some embodiments the target site is on chromosome 2. In some embodiments the target site is on chromosome 2 at q37.3. In some embodiments, the target site is within PD-1. In some embodiments, the target site is within exon 3 of PD-1. In some embodiments, the target site is within exon 5 of PD-1. In some embodiments, the target site is within exon 2 of PD-1. In some embodiments, the target site is one of the target sites listed in Table 5. In some embodiments, the gRNA that targets the target site comprises the SEQ ID NO of any that are listed in Table 5. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chr2:241, 852, 265-241, 852, 284. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chr2:241, 851, 154-241, 851, 173. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chr2:241,852, 752- 241,852,771. In some embodiments the target site is at chromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates chr2:241, 852, 729-241, 852, 748. In some embodiments the target site is atchromosome 16 and is within 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp or 20 bp of genomic coordinates ch2:241, 852, 672-241, 852, 691.

[0375] In some embodiments, the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence selected from SEQ ID NOs: 8-12. In some embodiments, the spacer of the gRNA is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence selected from SEQ ID NO: 30-34. In some embodiments, the spacer of the gRNA is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence as set forth in SEQ ID NO: 8. In some embodiments, the spacer of the gRNA is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence as set forth in SEQ ID NO: 10.

[0376] In some embodiments, the target site is adjacent to a protospacer adjacent motif (PAM). In some embodiments, the PAM sequence is or comprises “NGG” or is or comprises SEQ ID NO: 47. In some embodiments, the PAM sequence is or comprises “NNGRRT” or is or comprises SEQ ID NO: 48. In some embodiments, the gRNA comprises a scaffold for binding to a Cas protein with a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of the sequence set forth in SEQ ID NO: 45.Table 5

[0377] In some embodiments, the spacer of the gRNA is attached to a scaffold sequence. In some embodiments, the scaffold binds to a Cas protein. In some embodiments, the spacer is part of a gRNA and binds the gRNA to a Cas protein. In some embodiments, the scaffold is a sequence which binds to spCas9. In some embodiments, the scaffold comprises a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence as set forth in SEQ ID NO: 46. In some embodiments, the scaffold is encoded in DNA and has a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence as set forth in SEQ ID NO: 45.

[0378] In some embodiments, the spacer of the gRNA is attached to a scaffold sequence. In some embodiments, the scaffold binds to a Cas protein. In some embodiments, the spacer is part of a gRNA and binds the gRNA to a Cas protein. In some embodiments, the scaffold is a sequence which binds to MAD7. In some embodiments, the scaffold comprises a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a sequence as set forth in SEQ ID NO: 50. In some embodiments, the scaffold comprises any of the scaffolds described in US9,982,279, which is hereby incorporated in its entirety.2. Genetic Modifying agents

[0379] Methods for generating a genetic modification or disruption, including those described herein, can involve the use of one or more agent(s) capable of inducing a genetic modification or disruption, such as engineered systems to induce a genetic modification or disruption, a cleavage and / or a double strand break (DSB) or a nick in a target site or target position in the endogenous DNA such that repair of the break by an error born process such as non-homologous end joining (NHEJ). In some aspects, the methods for generating the genetically modified cells involve introducing a genetic modification or disruption at or near a target site, such as those described in Section I-D-l.

[0380] In some aspects, the genetic modification or disruption is introduced using a CRISPR-Cas combination comprising a guide RNA (gRNA), such as those described in Section I-D-l above, and a Cas protein.

[0381] . In some embodiments, the agent comprises a CRISPR / Cas that specifically binds to, recognizes, or hybridizes to the target site(s). In some embodiments, the CRISPR / Cas system includes an engineered crRNA / tracr RNA (“single guide RNA”) to guide specific cleavage. In some embodiments, the agent comprises nucleases based on the Argonaute system (e.g., from T. thermophilus, known as ‘TtAgo’, (Swarts et al. (2014) Nature 507(7491): 258- 261). Targeted cleavage using any of the nuclease systems described herein can be exploited to modify or disrupt a gene, e.g., knock out a gene with a target site located within it that the gRNA binds to.

[0382] In some aspects, the DNA-binding domain comprises a CRISPR-associated (Cas) protein or variant thereof, or is derived from a Cas protein or variant thereof.

[0383] In some embodiments, the Cas protein is derived from a Class 1 CRISPR system (i.e. multiple Cas protein system), such as a Type I or Type III system. In some embodiments, the Cas protein is derived from a Class 2 CRISPR system (i.e. single Cas protein system), such as a Type II, Type V, or Type VI CRISPR system. In some embodiments, the Cas protein is from a Type V CRISPR system. In some embodiments, the Cas protein is derived from a Cas 12 protein (i.e. Cpfl) or variant thereof, for example as described in WO 2017 / 189308 and Zetsche, B. et al. Cell. 163(3):759-71 (2015). In some embodiments, the Cas protein is derived from a Type II CRISPR system. In some embodiments, the Cas protein is derived from a Cas9 protein or variant thereof, for example as described in WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2014 / 093655, Jinek, M. et al. Science 337(6096):816-21 (2012), Mali, P. et al. Science 339(6121):823-6 (2013), Cong, L. et al. Science 339(6121):819-23 (2013), Perez- Pinera, P. et al. Nat. Methods 10, 973-976 (2013), or Mali, P. et al. Nat. Biotechnol. 31, 833- 838 (2013). Various CRISPR / Cas systems and associated Cas proteins for use in gene editing and regulation have been described, for example in Moon, S.B. et al. Exp. Mol. Med. 51, 1-11 (2019), Zhang, F. Q. Rev. Biophys. 52, E6 (2019), and Makarova K.S. et al. Methods Mol. Biol. 1311:47-75 (2015).

[0384] In some embodiments, the Cas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule, or variant thereof. In some embodiments, the Cas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule of .S'. pyogenes, S.thermophilus, S. aureus, C. jejuni, N. meningitidis, F. novicida, S. canis, S. auricularis, or variant thereof. In some embodiments, the Cas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of .S'. aureus. In some embodiments, the Cas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of .S', pyogenes.

[0385] Non-limiting examples of Cas9 orthologs from other bacterial strains include but are not limited to: Cas proteins identified in Acaryochloris marina MBIC 11017; Acetohalobium arabaticum DSM 5501; Acidithiobacillus caldus; Acidithiobacillus ferrooxidans ATCC 23270; Alicyclobacillus acidocaldarius LAA1; Alicyclobacillus acidocaldarius subsp. acidocaldarius DSM 446; Allochromatium vinosum DSM 180; Ammonifex degensii KC4; Anabaena variabilis ATCC 29413; Arthrospira maxima CS-328; Arthrospira platensis str. Paraca; Arthrospira sp. PCC 8005; Bacillus pseudomycoides DSM 12442; Bacillus selenitireducens MLS 10; Burkholderiales bacterium 1_1_47; Caldicelulosiruptor becscii DSM 6725; Candidatus Desulforudis audaxviator MP104C; Caldicellulosiruptor hydrothermalis 108; Clostridium phage c-st; Clostridium botulinum A3 str. Loch Maree; Clostridium botulinum Ba4 str. 657; Clostridium difficile QCD-63q42; Crocosphaera watsonii WH 8501; Cyanothece sp. ATCC 51142; Cyanothece sp. CCY0110; Cyanothece sp. PCC 7424; Cyanothece sp. PCC 7822; Exiguobacterium sibiricum 255-15; Finegoldia magna ATCC 29328; Ktedonobacter racemifer DSM 44963; Lactobacillus delbrueckii subsp. bulgaricus PB2003 / 044-T3-4; Lactobacillus salivarius ATCC 11741; Listeria innocua; Lyngbya sp. PCC 8106; Marinobacter sp. ELB 17; Methanohalobium evestigatum Z-7303; Microcystis phage Ma-LMMOl; Microcystis aeruginosa NIES-843; Microscilla marina ATCC 23134; Microcoleus chthonoplastes PCC 7420; Neisseria meningitidis; Nitrosococcus halophilus Nc4; Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111; Nodularia spumigena CCY9414; Nostoc sp. PCC 7120; Oscillatoria sp. PCC 6506; Pelotomaculum_thermopropionicum SI; Petrotoga mobilis SJ95; Polaromonas naphthalenivorans CJ2; Polaromonas sp. JS666; Pseudoalteromonas haloplanktis TAC125; Streptomyces pristinaespiralis ATCC 25486; Streptomyces pristinaespiralis ATCC 25486; Streptococcus thermophilus; Streptomyces viridochromogenes DSM 40736; Streptosporangium roseum DSM 43021; Synechococcus sp. PCC 7335; and Thermosipho africanus TCF52B (Chylinski et al., RNA Biol., 2013; 10(5): 726-737).

[0386] In some embodiments, the Cas9 protein or a variant thereof is derived from a Staphylococcus aureus Cas9 (SaCas9) protein or a variant thereof.

[0387] In some embodiments, the Cas9 protein or variant thereof is derived from aStreptococcus pyogenes Cas9 (SpCas9) protein or a variant thereof. In some embodiments, the spCas9 protein has at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of the amino acid sequence set forth in SEQ ID NOs: 49. In some embodiments, the Cas protein binds to a scaffold sequence that has at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of the nucleic sequence set forth in SEQ ID NOs: 46.

[0388] In some embodiments, the Cas protein is derived from a class 2 CRISPR system. In some embodiments, the Cas protein is derived from a class 2 type V-A CRISPR system. In some embodiments, the Cas protein is derived from a Casl2a / Cpfl protein or variant thereof. In some embodiments, the Cas protein is derived from a Cas protein isolated from Eubacterium rectale. In some embodiments, the Cas protein is MAD7. In some embodiments, the Cas protein has at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of the amino acid sequence set forth in SEQ ID NOs: 51. In some embodiments, the Cas protein is encoded by a nucleic acid sequence which has at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of the sequence set forth in SEQ ID NOs: 52. In some embodiments, the Cas protein binds to a scaffold sequence that has at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of the nucleic sequence set forth in SEQ ID NOs: 50.3. Delivery of Agents

[0389] In some embodiments, the genetic disruption, is carried out by delivering or introducing one or more agent(s), such as a first agent and / or a second agent, capable of inducing a genetic disruption, e.g., Cas9 and / or gRNA components, to a cell, using any of a number of known delivery method or vehicle for introduction or transfer to cells, for example, using viral delivery vectors, or any of the known methods or vehicles for delivering Cas molecules and gRNAs. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505. In some embodiments, nucleic acid sequences encoding one or more components of one or more agent(s) capable of inducing a genetic disruption is introduced into the cells, e.g., by anymethods for introducing nucleic acids into a cell described herein or known. In some embodiments, a vector encoding components of one or more agent(s) capable of inducing a genetic disruption such as a CRISPR guide RNA and / or a Cas enzyme can be delivered into the cell.

[0390] In some embodiments, the one or more agent(s) capable of inducing a genetic disruption, e.g., one or more agent(s) that is a Cas / gRNA, is introduced into the cell as a ribonucleoprotein (RNP) complex. RNP complexes include a sequence of ribonucleotides, such as an RNA or a gRNA molecule, and a protein, such as a Cas protein or variant thereof. For example, the Cas protein is delivered as RNP complex that comprises a Cas protein and a gRNA molecule targeting the target sequence, e.g., using electroporation or other physical delivery method. In some embodiments, the RNP is delivered into the cell via electroporation or other physical means, e.g., particle gun, Calcium Phosphate transfection, cell compression or squeezing. In some embodiments, the RNP can cross the plasma membrane of a cell without the need for additional delivery agents (e.g., small molecule agents, lipids, etc.). In some embodiments, delivery of the one or more agent(s) capable of inducing genetic disruption, e.g., CRISPR / Cas, as an RNP offers an advantage that the targeted disruption occurs transiently, e.g., in cells to which the RNP is introduced, without propagation of the agent to cell progenies. For example, delivery by RNP minimizes the agent from being inherited to its progenies, thereby reducing the chance of off-target genetic disruption in the progenies. In such cases, the genetic disruption and the integration of transgene can be inherited by the progeny cells, but without the agent itself, which may further introduce off-target genetic disruptions, being passed on to the progeny cells.

[0391] Agent(s) and components capable of inducing a genetic disruption, e.g., a Cas molecule and gRNA molecule, can be introduced into target cells in a variety of forms using a variety of delivery methods and formulations, as set forth in Tables 5 and 6, or methods described in, e.g., WO 2015 / 161276; US 2015 / 0056705, US 2016 / 0272999, US 2017 / 0211075; or US 2017 / 0016027. As described further herein, the delivery methods and formulations can be used to deliver template polynucleotides and / or other agents to the cell (such as those required for modifying the cells) in prior or subsequent steps of the methods described herein. When a Cas or gRNA component is encoded as DNA for delivery, the DNA may typically but not necessarily include a control region, e.g., comprising a promoter, to effect expression. Exemplary promoters for Cas molecule sequences include, e.g., CMV, EFla, EFS, MSCV,PGK, or CAG promoters. Useful promoters for gRNAs include, e.g., Hl, EF-la, tRNA or U6 promoters. Promoters with similar or dissimilar strengths can be selected to tune the expression of components. Sequences encoding a Cas molecule may comprise a nuclear localization signal (NLS), e.g., an SV40 NLS. In some embodiments a promoter for a Cas molecule or a gRNA molecule may be, independently, inducible, tissue specific, or cell specific. In some embodiments, an agent capable of inducing a genetic disruption is introduced RNP complexes.Table 6. Exemplary Delivery MethodsTable 7. Comparison of Exemplary Delivery Methods

[0392] In some embodiments, DNA encoding Cas molecules and / or gRNA molecules, or RNP complexes comprising a Cas molecule and / or gRNA molecules, can be delivered into cells by known methods or as described herein. For example, Cas-encoding and / or gRNA-encoding DNA can be delivered, e.g., by vectors (e.g., viral or non-viral vectors), non-vector based methods (e.g., using naked DNA or DNA complexes), or a combination thereof. In some embodiments, the polynucleotide containing the agent(s) and / or components thereof is delivered by a vector (e.g., viral vector / virus or plasmid). The vector may be any described herein.

[0393] In some aspects, a CRISPR enzyme (e.g. Cas nuclease) in combination with (and optionally complexed with) a guide sequence is delivered to the cell. For example, one or more elements of a CRISPR system is derived from a type I, type II, type III, or type V CRISPR system. For example, one or more elements of a CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes, Staphylococcus aureus, Eubacterium rectale or Neisseria meningitides .

[0394] In some embodiments, a Cas9 nuclease (e.g., that encoded by mRNA from Staphylococcus aureus or from Streptococcus pyogenes, e.g. pCW-Cas9, Addgene #50661, Wang et al. (2014) Science, 3:343-80-4; or nuclease or nickase lentiviral vectors available from Applied Biological Materials (ABM; Canada) as Cat. No. K002, K003, K005 or K006) and a guide RNA specific to the target locus (e.g. SOCS1, C1SH, or PD-1 locus in humans) are introduced into cells. In some embodiments, a MAD7 nuclease and a guide RNA specific to thetarget locus (e.g. SOCS1, CISH, or PD-1 locus in humans) are introduced into cells.

[0395] In some embodiments, the polynucleotide containing the agent(s) and / or components thereof or RNP complex is delivered by a non- vector based method (e.g., using naked DNA or DNA complexes). For example, the DNA or RNA or proteins or combination thereof, e.g., ribonucleoprotein (RNP) complexes, can be delivered, e.g., by organically modified silica or silicate (Ormosil), electroporation, transient cell compression or squeezing (such as described in Lee, et al. (2012) Nano Lett 12: 6322-27, Kollmannsperger et al (2016) Nat Comm 7, 10372), gene gun, sonoporation, magnetofection, lipid- mediated transfection, dendrimers, inorganic nanoparticles, calcium phosphates, or a combination thereof.

[0396] In some embodiments, delivery via electroporation comprises mixing the cells with the Cas-and / or gRNA-encoding DNA or RNP complex in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude. In some embodiments, delivery via electroporation is performed using a system in which cells are mixed with the Cas-and / or gRNA-encoding DNA in a vessel connected to a device (e.g., a pump) which feeds the mixture into a cartridge, chamber or cuvette wherein one or more electrical impulses of defined duration and amplitude are applied, after which the cells are delivered to a second vessel.

[0397] In some embodiments, the delivery vehicle includes an amphiphilic peptide reagent to mediate intracellular delivery of the Cas-and / or gRNA encoding DNA or RNP complex. In some embodiments, the delivery vehicle includes an amphiphilic peptide allowing for peptide- enabled ribonucleoprotein delivery for CRISPR engineering (PERC). In some embodiments, the genetic engineering is PERC-mediated genetic engineering. In some embodiments, the amphiphilic peptide reagent and the RNP complex are mixed prior to contacting the TIL with the amphiphilic peptide reagent and the RNP complex mix. In some embodiments, the amphiphilic peptide reagent comprises INF7TAT-A5K (A5K) and / or INF7TAT-P55(P55). In some embodiments, the genetic engineering is carried out using any of the methods described in e.g. Sahu et al. (2024) Biorxiv DOI: 10.1101 / 2024.07.14.603391.

[0398] In some embodiments, the delivery vehicle is a non-viral vector. In some embodiments, the non-viral vector is an inorganic nanoparticle. Exemplary inorganic nanoparticles include, e.g., magnetic nanoparticles (e.g., Fe3MnO2) and silica. The outer surface of the nanoparticle can be conjugated with a positively charged polymer (e.g., polyethylenimine, poly lysine, poly serine) which allows for attachment (e.g., conjugation or entrapment) ofpayload. In some embodiments, the non-viral vector is an organic nanoparticle. Exemplary organic nanoparticles include, e.g., SNALP liposomes that contain cationic lipids together with neutral helper lipids which are coated with polyethylene glycol (PEG), and protamine-nucleic acid complexes coated with lipid. Exemplary lipids and polymers for gene transfer include those described in, for example, WO 2019 / 195492 and WO 2020 / 223535.

[0399] In some embodiments, the vehicle has targeting modifications to increase target cell update of nanoparticles and liposomes, e.g., cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars, and cell penetrating peptides. In some embodiments, the vehicle uses fusogenic and endosome-destabilizing peptides / polymers. In some embodiments, the vehicle undergoes acid-triggered conformational changes (e.g., to accelerate endosomal escape of the cargo). In some embodiments, a stimulus-cleavable polymer is used, e.g., for release in a cellular compartment. For example, disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.

[0400] In some embodiments, the delivery vehicle is a biological non-viral delivery vehicle. In some embodiments, the vehicle is an attenuated bacterium (e.g., naturally or artificially engineered to be invasive but attenuated to prevent pathogenesis and expressing the transgene (e.g., Listeria monocytogenes, certain Salmonella strains, Bifidobacterium longum, and modified Escherichia coll), bacteria having nutritional and tissue- specific tropism to target specific cells, bacteria having modified surface proteins to alter target cell specificity). In some embodiments, the vehicle is a genetically modified bacteriophage (e.g., engineered phages having large packaging capacity, less immunogenicity, containing mammalian plasmid maintenance sequences and having incorporated targeting ligands). In some embodiments, the vehicle is a mammalian virus-like particle. For example, modified viral particles can be generated (e.g., by purification of the “empty” particles followed by ex vivo assembly of the virus with the desired cargo). The vehicle can also be engineered to incorporate targeting ligands to alter target tissuespecificity. In some embodiments, the vehicle is a biological liposome. For example, the biological liposome is a phospholipid-based particle derived from human cells (e.g., erythrocyte ghosts, which are red blood cells broken down into spherical structures derived from the subject (e.g., tissue targeting can be achieved by attachment of various tissue or cell-specific ligands), or secretory exosomes -subject-derived membrane-bound nanovescicles (30 -100 nm) of endocytic origin (e.g., can be produced from various cell types and can therefore be taken up by cells without the need for targeting ligands).

[0401] In some embodiments, RNA encoding Cas molecules and / or gRNA molecules, can be delivered into cells, e.g., target cells described herein, by known methods or as described herein. For example, Cas-encoding and / or gRNA-encoding RNA can be delivered, e.g., by microinjection, electroporation, transient cell compression or squeezing (such as described in Lee, et al. (2012) Nano Lett 12: 6322-27), lipid-mediated transfection, peptide-mediated delivery, e.g., cell-penetrating peptides, or a combination thereof.

[0402] In some embodiments, delivery via electroporation comprises mixing the cells with the RNA encoding Cas molecules and / or gRNA molecules in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude. In some embodiments, delivery via electroporation is performed using a system in which cells are mixed with the RNA encoding Cas molecules and / or gRNA molecules in a vessel connected to a device (e.g., a pump) which feeds the mixture into a cartridge, chamber or cuvette wherein one or more electrical impulses of defined duration and amplitude are applied, after which the cells are delivered to a second vessel.

[0403] In some embodiments, Cas molecules can be delivered into cells by known methods or as described herein. For example, Cas protein molecules can be delivered, e.g., by microinjection, electroporation, transient cell compression or squeezing (such as described in Lee, et al. (2012) Nano Lett 12: 6322-27), lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. Delivery can be accompanied by DNA encoding a gRNA or by a gRNA.

[0404] In some embodiments, the one or more agent(s) capable of introducing a cleavage, e.g., a Cas / gRNA system, is introduced into the cell as a ribonucleoprotein (RNP) complex. RNP complexes include a sequence of ribonucleotides, such as an RNA or a gRNA molecule, and a protein, such as a Cas protein or variant thereof. For example, the Cas protein is delivered as RNP complex that comprises a Cas protein and a gRNA molecule targeting the target sequence, e.g., using electroporation or other physical delivery method. In some embodiments, the RNP is delivered into the cell via electroporation or other physical means, e.g., particle gun, calcium phosphate transfection, cell compression or squeezing.

[0405] In some embodiments, delivery via electroporation comprises mixing the cells with the Cas9 molecules with or without gRNA molecules in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude. In some embodiments, delivery via electroporation is performed using a system in which cells are mixedwith the Cas9 molecules with or without gRNA molecules in a vessel connected to a device (e.g., a pump) which feeds the mixture into a cartridge, chamber or cuvette wherein one or more electrical impulses of defined duration and amplitude are applied, after which the cells are delivered to a second vessel.

[0406] In some embodiments, delivery via electroporation comprises mixing the cells with the Cas molecules with or without gRNA molecules in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude. In some embodiments, delivery via electroporation is performed using a system in which cells are mixed with the Cas molecules.

[0407] In some embodiments, the polynucleotide containing the agent(s) and / or components thereof is delivered by a combination of a vector and a non- vector based method. For example, a virosome comprises a liposome combined with an inactivated virus (e.g., HIV or influenza virus), which can result in more efficient gene transfer than either a viral or a liposomal method alone.

[0408] In some embodiments, the one or more agent(s) is or comprises a ribonucleoprotein (RNP) complex. In some embodiments, the concentration of the RNP incubated with, added to or contacted with the cells for modification is at a concentration of at or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40 or 50 pM, or a range defined by any two of the foregoing values. In some aspects, the concentration of the RNP is between at or about 1 pM and at or about 5 pM. In some embodiments, the concentration of the RNP incubated with, added to or contacted with the cells for modification is at a concentration of at or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 4 or 5 pM, or a range defined by any two of the foregoing values. In some aspects, the concentration of the RNP is between at or about 1.5 pM and at or about 2.5 pM. In some embodiments, the concentration of RNPs is at or about 2 pM.

[0409] In some aspects, the concentration of the RNP is between at or about 1 pM and at or about 5 pM, between at or about 1.5 pM and at or about 2.5 pM, between at or about 1.7 pM and at or about 2.5 pM, or between at or about 2 pM and at or about 2.5 pM. In some of any embodiments, the concentration of the RNP is at or about 1.0 pM, at or about 1.5 pM, at or about 1.7 pM, at or about 2 pM, at or about 2.2 pM, or at or about 2.5 pM, or a range defined by any two of the foregoing values. In some of any embodiments, the concentration of the RNP isat or about 2.0 |aM to at or about 2.5 pM. In some of any embodiments, the concentration of the RNP is at or about 1.7 |jM to at or about 2.5 pM. In some aspects, the concentration of the first RNP and / or the second RNP is between at or about 1 pM to at or about 5 pM. In some aspects, the concentration of the first RNP and / or the second RNP is at or about 1.5 pM. In some aspects, the concentration of the first RNP and / or the second RNP is at or about 1.7 pM. In some aspects, the concentration of the first RNP and / or the second RNP is at or about 2 pM. In some aspects, the concentration of the first RNP and / or the second RNP is at or about 2.2 pM. In some aspects, the concentration of the first RNP and / or the second RNP is at or about 2.5 pM.

[0410] In some embodiments, in the RNP complex, the ratio, e.g. the molar ratio, of the gRNA and the Cas molecule or other nucleases is at or about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4 or 1:5, or a range defined by any two of the foregoing values. In some embodiments, in the RNP complex, the ratio, e.g., molar ratio, of the gRNA and the Cas molecule or other nucleases is at or about 3:1, 2.9:1, 2.8:1, 2.7:1, 2.6:1, 2.5:1, 2.4:1, 2.3:1, 2.2:1, 2.1:1, 2:1 or 1:1, or a range defined by any two of the foregoing values.

[0411] In some embodiments, the polynucleotide is a linear or circular polynucleotide, such as a linear or circular DNA or linear RNA and can be delivered using any of the methods described in Section I.D herein (e.g., Tables 5 and 6) for delivering polynucleotides into the cell.

[0412] In particular embodiments, the polynucleotide, e.g., the template polynucleotide, are introduced into the cells in nucleotide form, e.g., as or within a non-viral vector. In some embodiments, the non-viral vector is or includes a polynucleotide, e.g., a DNA or RNA polynucleotide, that is suitable for transduction and / or transfection by any suitable and / or known non-viral method for gene delivery, such as but not limited to microinjection, electroporation, transient cell compression or squeezing (e.g., as described in Lee, et al. (2012) Nano Lett 12: 6322-27), lipid-mediated transfection, peptide-mediated delivery, e.g., cell-penetrating peptides, or a combination thereof. In some embodiments, the non-viral polynucleotide is delivered into the cell by a non-viral method described herein, such as a non-viral method listed in Table 7 herein.

[0413] In some embodiments, the polynucleotide sequence can be comprised in a vector molecule containing sequences that are not homologous to the region of interest in the genomic DNA.

[0414] In some embodiments, the polynucleotides and sequences encoding the one or moreagents may be on the same vector, for example an AAV vector. In some embodiments, the polynucleotides are delivered using an AAV vector and the one or more agents for inducing a genetic disruption, e.g., one or more CRISPR-Cas combination, are delivered as a different form, e.g., as mRNAs encoding the nucleases and / or gRNAs. In some embodiments, the polynucleotides and nucleases are delivered using the same type of method, e.g., a viral vector, but on separate vectors. In some embodiments, the polynucleotides are delivered in a different delivery system as the agents capable of inducing a genetic disruption, e.g., nucleases and / or gRNAs. In some embodiments, the polynucleotide is excised from a vector backbone in vivo, e.g., it is flanked by gRNA recognition sequences. In some embodiments, the polynucleotide is on a separate polynucleotide molecule as the Cas molecule and gRNA. In some embodiments, the Cas molecule and the gRNA are introduced in the form of a ribonucleoprotein (RNP) complex, and the polynucleotide is introduced as a polynucleotide molecule, e.g., in a vector or a linear polynucleotide, e.g., linear DNA. Types or nucleic acids and vectors for delivery include any of those described herein.E. Stimulation of T cells for Expansion (e.g. Second Expansion)

[0415] In some embodiments, the stimulation of T cells for expansion performed after genetically modifying the sorted cells is a second expansion. In some embodiments, the sorted cells are genetically modified without going through an expansion and the post-modification expansion is the first expansion. In some embodiments, the sorted cells are genetically modified without going through an expansion and there are two post-modification expansions (e.g. a first and second expansion after genetic engineering). In some embodiments, the stimulation of T cells for expansion is done before genetically modifying the cells (e.g. a first expansion) and after the cells have been genetically modified (e.g. a second expansion). In some embodiments, the sorted cells are genetically modified without going through an expansion and after the cells have been genetically modified, the cells are stimulated for expansion (e.g. a first expansion) and after the cells are stimulated for expansion, they are stimulated for expansion again (e.g. a second expansion). In some embodiments, the first and second expansion are identically performed. In some embodiments, the second expansion is performed differently from the first expansion. In some embodiments, the first expansion can be done utilizing any of the methods, reagents, or concentrations mentioned in this section. In some embodiments, the secondexpansion can be done utilizing any of the methods, reagents, or concentrations mentioned in this section.

[0416] The provided methods further include expanding the population of selected TILs by culture with one or more T-cell stimulating agent of lymphocytes under conditions to produce a population of expanded T cells. Hence, the provided methods involve the ex vivo expansion and production of a T cell therapeutic composition, particularly for use in connection with treating cancer. In some embodiments, the provided methods include a first and a second expansion of the population of selected TILs by culture with one or more T-cell simulating agents of lymphocytes under conditions to produce a population of expanded T cells. In some embodiments, the first expansion happens before the TILs are genetically modified and the second expansion happens after the TILs are genetically modified. In some embodiments, the TILs are genetically modified without an expansion, then after they TILs are genetically modified, the TILs go through a single expansion.

[0417] In some embodiments, the incubation or culturing with one or more T cell stimulatory agent(s) results in expansion or outgrowth of selected T cells, or a desired subset or subtype thereof or for viable cells thereof, for use in subsequent steps of the provided methods. Non-limiting examples of T cell stimulatory agent(s) and conditions for incubation or culture are described herein.

[0418] Thus, among the provided methods are methods of culturing T cells for manufacture of tumor reactive T cells in which T cells are cultured or incubated in the presence of a T cell stimulatory agent under conditions to expand T cells.

[0419] In some embodiments, the T cell stimulatory agent(s) include a recombinant T cell stimulating cytokine, such as IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27 and / or IL-35. In some embodiments, the T cell stimulating cytokine includes IL-2, alone or in combination with another cytokine from among IL-7, IL- 15, IL-21, IL-25, IL-23, IL- 27 and / or IL-35. In some embodiments, the T cell stimulatory agent(s) include a recombinant T cell stimulating cytokine, such as IL-2, IL-7, IL-15, IL-21, IL-25 and / or IL-23. In some embodiments, the T cell stimulating cytokine includes IL-2, alone or in combination with another cytokine from among IL-7, IL- 15, IL-21, IL-25 and / or IL-23. In some embodiments, the T cell stimulating cytokine is one, two, three or more of IL-2, IL-7, IL- 15 and IL-21. In some embodiments, the T cell stimulating cytokine includes IL-2, alone or in combination with another cytokine from among IL-7, IL- 15, and / or IL-21. In some embodiments, the T cell stimulating cytokine includes IL-2,alone or in combination with another cytokine from IL-25, IL-23, IL-27 and / or IL-35. In some embodiments, the T cell stimulating cytokines are IL-7 and IL- 15.

[0420] In some embodiments, the choice of cytokine or combination of cytokines is within the level of a skilled artisan, so long as the cytokines or cytokines provide activity to stimulate the T cells to expand. The activity to stimulate tumor reactive T cells can be direct or indirect. In some embodiments, the one or more cytokines directly stimulate tumor reactive T cells to expand or proliferate. In some embodiments, the one or more cytokines suppress T regulatory T cells, thereby indirectly stimulating or enhancing proliferation of desired tumor reactive T cells. In some embodiments, the T-cell stimulating agents include anti-CD3 (e.g. anti-CD3 antibody, such as OKT3), anti-CD28 reagents (e.g. anti-CD28 antibody), such as an anti-CD3 antibody (e.g. OKT3) and an anti-CD28 antibody and / or one or more recombinant cytokine (e.g. IL-2, IL- 7, IL-21 and / or IL- 15) . In some embodiments, the T-cell simulating agents include anti-CD3 antibody but not an anti-CD28 antibody.

[0421] In some embodiments, the incubation with a T cell stimulatory agent(s) for the initial expansion does not include incubation with an agent or agents that engage CD3 and a costimulatory molecule, such as CD28. In some embodiments, the incubation with a T cell stimulatory agent(s) for the initial expansion does not include incubation with an anti-CD3 antibody, such as OKT3. In some embodiments, the incubation with a T cell stimulatory agent(s) for the initial expansion does not include incubation with an anti-CD3 (e.g. OKT3) / anti- CD28 antibody, presented by APC’s, immobilized on a solid surface (e.g. bead), or as a soluble antibody. In some embodiment, the incubation with a T cell stimulatory agent(s) for the initial expansion does not include incubation with soluble anti-CD3, such as OKT3. In some embodiment, the incubation with a T cell stimulatory agent(s) for the initial expansion does not include incubation with an anti-CD3 / anti-CD28, including such reagents immobilized on beads, e.g. as provided by Dynabeads. In some embodiments, the incubation with a T cell stimulatory agent(s) for the initial expansion does not include incubation with APCs, such as irradiated APCs. In some embodiments, the incubation with a T cell stimulatory agent(s) for the initial expansion does not include incubation with non-dividing PBMCs, such as irradiated PBMCs.

[0422] In some of any of the provided embodiments, the T cell stimulatory agent(s) is selected from an agent that initiates TCR / CD3 intracellular signaling and an agent that initiates signaling via a costimulatory receptor. In some of any of the provided embodiments, the agent that initiates TCR / CD3 intracellular signaling is an anti-CD3 antibody, such as OKT3. In someof any of the provided embodiments, the agent that initiates signaling via a costimulatory receptor comprises peripheral blood mononuclear cells (PBMCs), optionally non-dividing or irradiated PBMCs. In some of any of the provided embodiments, the agent that initiates signaling via a costimulatory receptor is an anti-CD28 antibody. In some of any of the provided embodiments, the T cell stimulatory agent(s) is an anti-CD3 antibody and an anti-CD28 antibody that each are soluble. In particular embodiments, one or more recombinant cytokines also are present as additional T cell stimulatory agents during the incubation. In some embodiments, the incubation with a T cell stimulatory agent(s) include incubation with at least one T cell stimulating recombinant cytokine (e.g. recombinant IL-2, IL-7, IL-21, IL-15, IL-25, IL-23, IL- 27, and / or IL-35) and a further T cell stimulatory agent(s) that engage CD3 and / or a costimulatory molecule (e.g. CD28) on T cells.

[0423] In embodiments of the provided methods, the stimulating conditions include one or more agent, e.g., ligand, which turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell and / or a costimulatory signal in a T cell. Such agents can include antibodies, such as those specific for a TCR component, e.g., anti-CD3, and / or costimulatory receptor, e.g. anti- CD28 or anti-4- IBB. In some embodiments, such agents are added to the culture medium as soluble antibodies. In other embodiments, such agents are bound to solid support such as a bead. In some embodiments, the T cell stimulatory agent(s) includes anti-CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0424] An anti-CD3 antibody can include any antibody directed against or that can specifically bind the CD3 receptor on the surface of T cells, typically human CD3 on human T cells. Anti-CD3 antibodies include OKT3, also known as muromonab. Anti-CD3 antibodies also include the UHCTI clone, also known as T3 and CD3E. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab. The anti-CD3 antibody can be added as a soluble reagent or bound to a bead. In particular embodiments, the anti-CD3 antibody is soluble.

[0425] In particular embodiments, the T cell stimulatory agent(s) include an anti-CD3 antibody, which is added to the cell culture medium during the incubation. In some embodiments, the anti-CD3 antibody is added at a concentration ranging between at or about 0.1 ng / mL and 50 ng / mL, such between at or about 0.5 ng / mL and at or about 50 ng / mL, between at or about 0.5 ng / mL and at or about 30 ng / mL, between at or about 0.5 ng / mL and at or about 15 ng / mL, between at or about 0.5 ng / mL and at or about 5 ng / mL, between at or about 0.5 ng / mLand at or about 1 ng / mL, between at or about 1 ng / mL and at or about 50 ng / mL, between at or about 1 ng / mL and at or about 30 ng / mL, between at or about 1 ng / mL and at or about 15 ng / mL, between at or about 1 ng / mL and at or about 5 ng / mL, between at or about 5 ng / mL and at or about 50 ng / mL, between at or about 5 ng / mL and at or about 30 ng / mL, between at or about 5 ng / mL and at or about 15 ng / mL, between at or about 15 ng / mL and at or 50 ng / mL, between at or about 15 ng / mL and at or about 30 ng / mL or between at or about 30 ng / mL and at or about 50 ng / mL, each inclusive.

[0426] In particular embodiments, the anti-CD3 antibody is OKT3. In an embodiment, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 pg / mL of OKT3 antibody. In an embodiment, the cell culture medium comprises between 0.1 ng / mL and 1 ng / mL, between 1 ng / mL and 5 ng / mL, between 5 ng / mL and 10 ng / mL, between 10 ng / mL and 20 ng / mL, between 20 ng / mL and 30 ng / mL, between 30 ng / mL and 40 ng / mL, between 40 ng / mL and 50 ng / mL, and between 50 ng / mL and 100 ng / mL of OKT3 antibody.

[0427] In some embodiments, the T cell stimulatory agent(s) includes incubation with an anti-CD3 antibody and incubation with a further agent that specifically binds to CD28 or stimulates or induces a CD28-mediated signal in cells. In some embodiments, the CD28- mediated signal can be initiated or provided by anti-CD28 antibody or antigen-binding fragment thereof. In some embodiments, the CD28-mediated signal can be provided by antigen- presenting feeder cells (APCs), such as peripheral blood mononuclear cells (PBMC).

[0428] In some embodiments, the T cell stimulatory agent(s) can include adding to the population of T cells feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC). In some aspects, the non-dividing feeder cells can comprise gamma- irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells. In some embodiments, the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded. In some embodiments, the ratio of T cells to PBMCs and / or antigen-presenting cells is about 1 to 25, about 1 to 50, about 1 to100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500.

[0429] In some embodiments, the stimulation does not include incubation with PBMCs or other feeder cells, such as non-divided or irradiated PBMCs or other non-dividing or irradiated feeder cells.

[0430] In some embodiments, the T cell stimulatory agent(s) can include adding to the population of cells an anti-CD28 antibody or antigen-binding fragment thereof. An anti-CD28 antibody can include any antibody directed against or that can specifically bind the CD28 receptor on the surface of T cells. Non-limiting examples of anti-CD28 antibodies include NA / LE (e.g. BD Pharmingen), IM1376 (e.g. Beckman Coulter), or 15E8 (e.g. Miltenyi Biotec). The anti-CD28 antibody can be added as a soluble reagent or bound to a bead. In particular embodiments, the anti-CD3 antibody is soluble. In some embodiments, the anti-CD28 antibody is added at a concentration ranging between at or about 1 ng / mL and 1000 ng / mL, between at or about 1 ng / mL and 500 ng / mL, between at or about 1 ng / mL and at or about 100 ng / mL, between at or about 1 ng / mL and at or about 10 ng / mL, between at or about 10 ng / mL and at or about 1000 ng / mL, between at or about 10 ng / mL and at or about 500 ng / mL, between at or about 10 ng / mL and at or about 100 ng / mL, between at or about 100 ng / mL and at or about 1000 ng / mL, between at or about 100 ng / mL and at or about 500 ng / mL or between at or about 500 ng / mL and at or about 1000 ng / mL.

[0431] In some embodiments, the T cell stimulatory agent(s) include one or more recombinant cytokine. In some embodiments, the cytokine is added or is exogenous to the culture media. Thus, in some embodiments, one or more further recombinant cytokine also is included during the culturing. In some embodiments, the recombinant cytokine can include one or more of IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27 and / or IL-35. In some embodiments, the recombinant cytokine can include one or more of IL-2, IL-7, IL- 15, IL-21, IL-25 and / or IL- 23. In some embodiments, the culturing and incubation is carried out in the presence of recombinant IL-2, IL- 15 and IL-7. In some embodiments, the culturing is carried out in the presence of a IL-2. In some embodiments, the culturing is carried out in the presence of IL- 15 and IL- 17, which, in some aspects does not additionally include IL-2. In particular embodiments, the recombinant cytokine(s) is human.

[0432] The recombinant cytokine generally is a recombinant human protein. In particular embodiments, the recombinant cytokine is present in the cell culture medium during the incubation at a concentration of at least or at least about 0.5 lU / mL, at least or at least about 1.0 lU / mL, at least or at least about 5 lU / mL, at least at or about or at or about 10 lU / mL, at least at or about or at or about 100 lU / mL, at least at or about or at or about 1000 lU / mL, at least at or about or at or about 1500 lU / mL, at least at or about or at or about 2000 lU / mL, at least at or about or at or about 2500 lU / mL, at least at or about or at or about 3000 lU / mL, at least at or about or at or about 3500 lU / mL, at least at or about or at or about 4000 lU / mL, at least at or about or at or about 4500 lU / mL, at least at or about or at or about 5000 lU / mL, at least at or about or at or about 5500 lU / mL, at least at or about or at or about 6000 lU / mL, at least at or about or at or about 6500 lU / mL, at least at or about or at or about 7000 lU / mL, at least at or about or at or about 7500 lU / mL, or at least at or about or at or about 8000 lU / mL. In an embodiment, the cell culture medium comprises between at or about 10 lU / mL and at or about 100 lU / mL, at or about 100 lU / mL and at or about 1000 lU / mL, at or about 1000 and at or about 2000 lU / mL, between at or about 2000 and at or about 3000 lU / mL, between at or about 3000 and 4000 at or about lU / mL, between at or about 4000 and at or about 5000 lU / mL, between at or about 5000 and at or about 6000 lU / mL, between at or about 6000 and at or about 7000 lU / mL, between at or about 7000 and at or about 8000 lU / mL, each inclusive.

[0433] In some embodiments, recombinant IL-2 is present in the cell culture medium. In some aspects, IL-2 is the only recombinant cytokine added to the culture. In some aspects, recombinant IL-2 and one other recombinant modulatory cytokine from IL-7, IL- 15, IL-21, IL- 23, IL-25, IL- 27 or IL-35 is added to the culture. IL-2 is a cytokine that supports T cell recovery and proliferation. IL-2 also supports the homeostasis of T cells, thereby supporting their phenotype, differentiation status, and immune memory. In some cases, induction of regulatory T cells in the tumor microenvironment may lead to low bioavailability of IL-2. Recombinant IL-2 has been regularly used in broad expansion of T cells in various contexts. Recombinant IL-2 is commercially available. In particular embodiments, recombinant IL-2 is GMP grade (e.g. MACS GMP Recombinant Human IL-2, Miltenyi Biotec).

[0434] Recombinant IL-2 can be included in cell culture media during various stages of the provided process. In some cases, recombinant IL-2 can be included in the initial T cell expansion (first expansion), such as to promote TIL outgrowth

[0435] In some embodiments, recombinant IL-2 is added to the culture medium at a concentration between at or about 10 lU / mL and at or about 1000 lU / mL, such as between at or about 10 lU / mL and at or about 600 lU / mL, between at or about 10 lU / mL and at or about 400 lU / mL, between at or about 10 lU / mL and at or about 200 lU / mL, between at or about 10 lU / mL and at or about 100 lU / mL, between at or about 10 lU / mL and at or about 50 lU / mL, between at or about 50 lU / mL and at or about 1000 lU / mL, between at or about 50 lU / mL and at or about 600 lU / mL, between at or about 50 lU / mL and at or about 400 lU / mL, between at or about 50 lU / mL and at or about 200 lU / mL, between at or about 50 lU / mL and at or about 100 lU / mL, between at or about 100 lU / mL and at or about 1000 lU / mL, between at or about 100 lU / mL and at or about 600 lU / mL, between at or about 100 lU / mL and at or about 400 lU / mL, between at or about 100 lU / mL and at or about 200 lU / mL, between at or about 200 lU / mL and at or about 1000 lU / mL, between at or about 200 lU / mL and at or about 600 lU / mL, between at or about 200 lU / mL and at or about 400 lU / mL, between at or about 400 lU / mL and at or about 1000 lU / mL, between at or about 400 lU / mL and at or about 600 lU / mL or between at or about 600 lU / mL and at or about 1000 lU / mL. In some embodiments, recombinant IL-2 is present in an amount that is between 50 and 400 lU / mL. In some embodiments, recombinant IL-2 is present in an amount that is between 500 and 6,000 lU / mL. In some embodiments, recombinant IL-2 is present in an amount that is between 1,000 and 5,000 lU / mL. In some embodiments, recombinant IL-2 is present in an amount that is between 2,000 and 4,000 lU / mL. In some embodiments, recombinant IL-2 is present in an amount that is at or about 3,000 lU / mL.

[0436] In some embodiments, the expansion is carried out in the presence of recombinant IL-2 added at a concentration of between 200 lU / mL and at or about 5000 lU / mL. In some embodiments, recombinant IL-2 Is added to the culture medium at a concentration of at or about 200 lU / mL, at or about 300 lU / mL, at or about 400 lU / mL, at or about 500 lU / mL, at or about 600 lU / mL, at or about 700 lU / mL, at or about 800 lU / mL, at or about 900 lU / mL, at or about 1000 lU / mL, or any concentration between any of the foregoing. In some embodiments, recombinant IL-2 Is added to the culture medium at a concentration of at or about 300 lU / mL. In some embodiments, recombinant IL-2 is added to the culture medium at a concentration of at or about 600 lU / mL. In some embodiments, recombinant IL-2 is added to the culture medium at a concentration of at or about 1000 lU / mL. In some embodiments, at least one other recombinant modulatory cytokine from IL-7, IL-15, IL-21, IL-23, IL-25, IL-27 or IL-35 is added to the culture medium.

[0437] In some embodiments, the incubation is carried out with a higher dose IL-2. In some aspects, IL-2 is the only recombinant cytokine added to the culture.

[0438] In some embodiments, the recombinant IL-2 is added to the culture medium at a concentration between at or about 1000 lU / mL at or about 8000 lU / mL, such as between at or about 1000 lU / mL and at or about 7000 lU / mL, between at or about 1000 lU / mL and at or about 6000 lU / mL, between at or about 1000 lU / mL and at or about 5000 lU / mL, between at or about 1000 lU / mL and at or about 4000 lU / mL, between at or about 1000 lU / mL and at or about 2000 lU / mL, 2000 lU / mL at or about 8000 lU / mL, between at or about 2000 lU / mL and at or about 7000 lU / mL, between at or about 2000 lU / mL and at or about 6000 lU / mL, between at or about 2000 lU / mL and at or about 5000 lU / mL, between at or about 2000 lU / mL and at or about 4000 lU / mL, 4000 lU / mL at or about 8000 lU / mL, between at or about 4000 lU / mL and at or about 7000 lU / mL, between at or about 4000 lU / mL and at or about 6000 lU / mL, between at or about 4000 lU / mL and at or about 5000 lU / mL, between at or about 5000 lU / mL at or about 8000 lU / mL, between at or about 5000 lU / mL and at or about 7000 lU / mL, between at or about 5000 lU / mL and at or about 6000 lU / mL, between at or about 6000 lU / mL at or about 8000 lU / mL, between at or about 6000 lU / mL and at or about 7000 lU / mL or between at or about 7000 lU / mL and at or about 8000 lU / mL. In some embodiments, recombinant IL-2 is present in an amount that is or is about 3000 lU / mL.

[0439] In some embodiments, recombinant IL- 15 is present in the cell culture medium. IL- 15 is a cytokine that is involved in memory T cell homeostasis and activation. In some cases, IL- 15 can promote effector functions of antigen-experienced T cells in the absence of antigen and prevent their differentiation into an exhausted phenotype. IL- 15 also plays a role in T cell proliferation. Recombinant IL- 15 is commercially available. In particular embodiments, recombinant IL-15 is GMP grade (e.g. MACS GMP Recombinant Human IL-15, Miltenyi Biotec).

[0440] Recombinant IL- 15 can be included in cell culture media during various stages of the provided process. In some cases, recombinant IL-15 can also be included in cultures to expand tumor-reactive T cells during the expansion phase. In some cases, recombinant IL- 15 can be combined with recombinant IL-7 to provide for activation, survival and / or expansion of tumor- reactive T cells in the provided methods. In some such embodiments, the combination of recombinant IL-7 and IL- 15 is an alternative to the use of recombinant IL-2 in the culture, and the culture media does not additionally contain recombinant IL-2.

[0441] In some embodiments, the recombinant IL- 15 is added to the culture medium at a concentration between at or about 10 lU / mL and 500 lU / mL, such as between at or about 10 lU / mL and at or about 400 lU / mL, between at or about 10 lU / mL and at or about 300 lU / mL, between at or about 10 lU / mL and at or about 200 lU / mL, between at or about 10 lU / mL and at or about 100 lU / mL, between at or about 10 lU / mL and at or about 70 lU / mL, between at or about 10 lU / mL and at or about 50 lU / mL, between at or about 10 lU / mL and at or about 30 IU / mL, between at or about 30 lU / mL and 500 lU / mL, between at or about 30 lU / mL and at or about 400 lU / mL, between at or about 30 lU / mL and at or about 300 lU / mL, between at or about 30 lU / mL and at or about 200 lU / mL, between at or about 30 lU / mL and at or about 100 lU / mL, between at or about 30 lU / mL and at or about 70 lU / mL, between at or about 30 lU / mL and at or about 50 lU / mL, between at or about 50 lU / mL and at or about 400 lU / mL, between at or about 50 lU / mL and at or about 500 lU / mL, between at or about 50 lU / mL and at or about 300 lU / mL, between at or about 50 lU / mL and at or about 200 lU / mL, between at or about 50 lU / mL and at or about 100 lU / mL, between at or about 50 lU / mL and at or about 70 lU / mL, between at or about 70 lU / mL and at or about 500 lU / mL, between at or about 70 lU / mL and at or about 400 lU / mL, between at or about 70 lU / mL and at or about 300 lU / mL, between at or about 70 lU / mL and at or about 200 lU / mL, between at or about 70 lU / mL and at or about 100 lU / mL, between at or about 100 lU / mL and at or about 500 lU / mL, between at or about 100 lU / mL and at or about 400 lU / mL, between at or about 100 lU / mL and at or about 300 lU / mL, between at or about 100 lU / mL and at or about 200 lU / mL, between at or about 200 lU / mL and at or about 500 lU / mL, between at or about 200 lU / mL and at or about 400 lU / mL, between at or about 200 lU / mL and at or about 300 lU / mL, between at or about 300 lU / mL and at or about 500 lU / mL, between at or about 200 lU / mL and at or about 400 lU / mL, or between at or about 400 lU / mL and at or about 500 lU / mL. In some embodiments, the IL-15 is added to the culture medium in an amount between at or about 100 lU / mL and at or about 200 lU / mL. In some embodiments, the IL- 15 is added to the culture medium at or about 500 lU / mL.

[0442] In some embodiments, the incubation is carried out with a higher dose IL- 15.

[0443] In some embodiments, the recombinant IL- 15 is added to the culture medium at a concentration between at or about 250 lU / mL and at or about 5000 lU / mL, such as between at or about 250 lU / mL and at or about 4000 lU / mL, between at or about 250 lU / mL and at or about 2000 lU / mL, between at or about 250 lU / mL and at or about 1500 lU / mL, between at or about 250 lU / mL and at or about 1000 lU / mL, between at or about 250 lU / mL and at or about 750lU / mL, between at or about 750 lU / mL and at or about 5000 lU / mL, between at or about 750 lU / mL and at or about 4000 lU / mL, between at or about 750 lU / mL and at or about 2000 lU / mL, between at or about 750 lU / mL and at or about 1500 lU / mL, between at or about 750 lU / mL and at or about 1000 lU / mL, between at or about 1000 lU / mL and at or about 5000 lU / mL, between at or about 1000 lU / mL and at or about 4000 lU / mL, between at or about 1000 lU / mL and at or about 2000 lU / mL, between at or about 1000 lU / mL and at or about 1500 lU / mL, between at or about 1500 lU / mL and at or about 5000 lU / mL, between at or about 1500 lU / mL and at or about 4000 lU / mL, between at or about 1500 lU / mL and at or about 2000 lU / mL, between at or about 2000 lU / mL and at or about 5000 lU / mL, such as between at or about 2000 lU / mL and at or about 4000 lU / mL, or between at or about 4000 lU / mL and at or about 5000 lU / mL. In some embodiments, the recombinant IL- 15 is added to the culture medium at a concentration between at or about 250 and at or about 1,500.

[0444] In some embodiments, the recombinant IL- 15 is added to the cell culture media at a concentration of at or about 500 lU / mL, at or about 600 lU / mL, at or about 700 lU / mL, at or about 800 lU / mL, at or about 900 lU / mL, at or about 1000 lU / mL, at or about 1100 lU / mL, at or about 1200 lU / mL, at or about 1300 lU / mL, at or about 1400 lU / mL, at or about 1500 lU / mL, at or about 1600 lU / mL, at or about 1700 lU / mL, at or about 1800 lU / mL, at or about 1900 lU / mL or at or about 2000 lU / mL, or any concentration between any of the foregoing. In some embodiments, IL- 15 is added to the culture medium at a concentration of at or about 1000 lU / mL.

[0445] In some embodiments, the initial expansion (e.g. first expansion) is carried out in the presence of recombinant IL- 15 added at a concentration of 500 lU / mL to 2000 lU / mL (e.g. at or about 1000 lU / mL). In some embodiments, the initial expansion (e.g. first expansion) is carried out in the presence of recombinant IL-15 added at a concentration of at or about 1000 lU / mL. In some embodiments, at least one other recombinant modulatory cytokine from IL-2, IL-7, IL-21, IL-23, IL-25, IL- 27 or IL-35 is added to the culture medium.

[0446] In some embodiments, recombinant IL- 15 and IL-2 are added to the culture medium. In some embodiments, recombinant IL- 15 is added at a concentration of 500 lU / mL to 2000 lU / mL (e.g. at or about 1000 lU / mL) and recombinant IL-2 is added at a concentration of 200 lU / mL to 5000 lU / mL (e.g. at or about 3000 lU / mL). In some embodiments, the expansion is carried out in the presence of recombinant IL- 15 added at 1000 lU / mL and recombinant IL-2added at 3000 lU / mL. In some embodiments, at least one other recombinant modulatory cytokine from IL-7, IL-21, IL-23, IL-25, IL- 27 or IL-35 is added to the culture medium.

[0447] In some embodiments, recombinant IL-7 is added to the culture medium. In some aspects, recombinant IL-7 is added to the culture media with one or both of IL-2 or IL- 15. In some aspects, recombinant IL-7 and recombinant IL-2 are added to the culture media. In some aspects, recombinant IL-7 and recombinant IL- 15 are added to the culture media. In some aspects, recombinant IL-7 (e.g. in combination with one or both of IL-2 and IL-15) and one other recombinant modulatory cytokine from IL-23, IL-25, IL- 27 or IL-35 is added to the culture medium. IL-7 is a cytokine that is involved in promoting T cell maintenance and homeostasis. In some cases, IL-7 can boost memory T cell survival and proliferation, particularly the central memory compartment. Recombinant IL-7 is commercially available. In particular embodiments, recombinant IL-7 is GMP grade (e.g. MACS GMP Recombinant Human IL-7, Miltenyi Biotec).

[0448] Recombinant IL-7 can be included in cell culture media during various stages of the provided process. In some cases, recombinant IL-7 can also be included in cultures to expand tumor-reactive T cells during the expansion phase. Inclusion of recombinant IL-7 in the process can maintain or support expansion of memory T cell subsets in the process. In some cases, recombinant IL-7 can be combined with recombinant IL- 15 to provide for activation, survival and / or expansion of tumor-reactive T cells in the provided methods. In some such embodiments, the combination of recombinant IL-7 and IL- 15 is an alternative to the use of recombinant IL-2 in the culture, and the culture media does not additionally contain recombinant IL-2.

[0449] In some embodiments, the recombinant IL-7 is added to the culture medium at a concentration between at or about 100 lU / mL and at or about 2000 lU / mL, between at or about 100 lU / mL and at or about 1500 lU / mL, between at or about 100 lU / mL and at or aboutlOOO lU / mL, between at or about 100 lU / mL and at or about 800 lU / mL, between at or about 100 lU / mL and at or about 600 lU / mL, between at or about 100 lU / mL and at or about 400 lU / mL, between at or about 100 lU / mL and at or about 200 lU / mL, between at or about 200 lU / mL and at or about 2000 lU / mL, between at or about 200 lU / mL and at or about 1500 lU / mL, between at or about 200 lU / mL and at or aboutlOOO lU / mL, between at or about 200 lU / mL and at or about 800 lU / mL, between at or about 200 lU / mL and at or about 600 lU / mL, between at or about 200 lU / mL and at or about 400 lU / mL, between at or about 400 lU / mL and at or about 2000 lU / mL, between at or about 400 lU / mL and at or about 1500 lU / mL, between at or about400 lU / mL and at or about 1000 lU / mL, between at or about 400 lU / mL and at or about 800 lU / mL, between at or about 400 lU / mL and at or about 600 lU / mL, between at or about 600 lU / mL and at or about 2000 lU / mL, between at or about 600 lU / mL and at or about 1500 lU / mL, between at or about 600 lU / mL and at or about 1000 lU / mL, between at or about 600 lU / mL and at or about 800 lU / mL, between at or about 800 lU / mL and at or about 2000 lU / mL, between at or about 800 lU / mL and at or about 1500 lU / mL, between at or about 800 lU / mL and at or about 1000 lU / mL, between at or about 1000 lU / mL and at or about 2000 lU / mL, between at or about 1000 lU / mL and at or about 1500 lU / mL, between at or about 1500 lU / mL and at or about 2000 lU / mL. In some embodiments, the IL-7 is added to the culture medium in an amount between at or about 1000 lU / mL and at or about 2000 lU / mL. In some embodiments, the IL-7 is added to the culture medium at or about 600 lU / mL. In some embodiments, IL-7 is added to the culture medium at or about 1000 lU / mL.

[0450] In some embodiments, recombinant IL-7 and IL-2 are added to the culture medium. In some embodiments, recombinant IL-7 is added at a concentration of 400 lU / mL to 2000 lU / mL (e.g. at or about 600 lU / mL or 1000 lU / mL) and recombinant IL-2 is added at a concentration of 200 lU / mL to 5000 lU / mL (e.g. at or about 3000 lU / mL). In some embodiments, the expansion is carried out in the presence of recombinant IL-7 added at 1000 lU / mL and recombinant IL-2 added at 3000 lU / mL. In some embodiments, the first expansion is carried out in the presence of recombinant IL-7 added at 600 lU / mL and recombinant IL-2 added at 3000 lU / mL. In some embodiments, at least one other recombinant modulatory cytokine from IL-15, IL-21, IL-23, IL-25, IL- 27 or IL-35 is added to the culture medium.

[0451] In some embodiments, recombinant IL- 15 and IL-7 are added to the culture medium. In some embodiments, recombinant IL- 15 is added at a concentration of 500 lU / mL to 2000 lU / mL (e.g. at or about 1000 lU / mL) and recombinant IL-7 is added at a concentration of 400 lU / mL to 2000 lU / mL (e.g. at or about 600 lU / mL or 1000 lU / mL). In some embodiments, the initial expansion (e.g. first expansion) is carried out in the presence of recombinant IL- 15 added at 1000 lU / mL and recombinant IL-7 added at 1000 lU / mL. In some embodiments, the first expansion is carried out in the presence of recombinant IL- 15 added at 1000 lU / mL and recombinant IL-7 added at 600 lU / mL. In some embodiments, at least one other recombinant modulatory cytokine from IL-2, IL-21, IL-23, IL-25, IL-27 or IL-35 is added to the culture medium.

[0452] In some embodiments, recombinant IL-21 is added to the culture medium. In some aspects, recombinant IL-21 is added to the culture media with one or both of IL-2, IL-7, or IL- 15. In some aspects, recombinant IL-21 and recombinant IL-2 are added to the culture media. In some aspects, recombinant IL-21 and recombinant IL- 15 are added to the culture media. In some aspects, recombinant IL-21 (e.g. in combination with one or more IL-2, IL-7 and IL- 15) and one other recombinant modulatory cytokine from IL-23, IL-25, IL-27 or IL-35 is added to the culture medium. IL-21 is a cytokine that supports a broad range of T cell activation without increasing regulatory T cell signaling. In some cases, IL-21 can support memory cell stabilization, effector function, and proliferation of antigen-experienced T cells. IL-21 can induce upregulation of effector molecules in both CD4 and CD8 T cells. Recombinant IL-21 is commercially available. In particular embodiments, recombinant IL-21 is GMP grade (e.g. MACS GMP Recombinant Human IL-21, Miltenyi Biotec).

[0453] Recombinant IL-21 can be included in cell culture media during various stages of the provided process. In some cases, recombinant IL-21 can also be included in cultures to expand tumor-reactive T cells during the expansion phase, such as to support proliferation and stabilization of memory phenotype. In some such embodiments, the combination of recombinant IL-21 and IL- 15 is an alternative to the use of recombinant IL-2 in the culture, and the culture media does not additionally contain recombinant IL-2.

[0454] In some embodiments, the recombinant IL-21 is added to the culture medium at a concentration between at or about 0.5 lU / mL and at or about 20 lU / mL, between at or about 0.5 lU / mL and at or about 15 lU / mL, between at or about 0.5 lU / mL and at or about 10 lU / mL, between at or about 0.5 lU / mL and at or about 5 lU / mL, between at or about 0.5 lU / mL and at or about 2.5 lU / mL, between at or about 0.5 lU / mL and at or about 1 lU / mL, between at or about 1 lU / mL and at or about 20 lU / mL, between at or about 1 lU / mL and at or about 15 lU / mL, between at or about 1 lU / mL and at or about 10 lU / mL, between at or about 1 lU / mL and at or about 5 lU / mL, between at or about 1 lU / mL and at or about 2.5 lU / mL, between at or about 2.5 lU / mL and at or about 20 lU / mL, between at or about 2.5 lU / mL and at or about 15 lU / mL, between at or about 2.5 lU / mL and at or about 10 lU / mL, between at or about 2.5 lU / mL and at or about 5 lU / mL, between at or about 5 lU / mL and at or about 20 lU / mL, between at or about 5 lU / mL and at or about 15 lU / mL, between at or about 5 lU / mL and at or about 10 lU / mL, between at or about 10 lU / mL and at or about 20 lU / mL, between at or about 10 lU / mL and at or about 15 lU / mL, or between at or about 15 lU / mL and at or about 20 lU / mL.In some embodiments, the IL-21 is added to the culture medium in an amount between at or about 0.5 lU / mL and at or about 2.5 lU / mL. In some embodiments, the IL-21 is added to the culture medium in an amount between at or about 0.63 lU / mL and at or about 10 lU / mL. In some embodiments, the IL-21 is added to the culture medium at or about 1 lU / mL. In some embodiments, the IL-21 is added to the culture medium at or about 1.15 lU / mL.

[0455] In some embodiments, the incubation is carried out with a higher dose IL-21.

[0456] In some embodiments, the recombinant IL-21 is added to the culture medium at a concentration between at or about 500 lU / mL and at or about 5000 lU / mL, such as between at or about 500 lU / mL and at or about 4000 lU / mL, between at or about 500 lU / mL and at or about 2000 lU / mL, between at or about 500 lU / mL and at or about 1500 lU / mL, between at or about 500 lU / mL and at or about 1000 lU / mL, between at or about 500 lU / mL and at or about 750 lU / mL, between at or about 750 lU / mL and at or about 5000 lU / mL, between at or about 750 lU / mL and at or about 4000 lU / mL, between at or about 750 lU / mL and at or about 2000 lU / mL, between at or about 750 lU / mL and at or about 1500 lU / mL, between at or about 750 lU / mL and at or about 1000 lU / mL, between at or about 1000 lU / mL and at or about 5000 lU / mL, between at or about 1000 lU / mL and at or about 4000 lU / mL, between at or about 1000 lU / mL and at or about 2000 lU / mL, between at or about 1000 lU / mL and at or about 1500 lU / mL, between at or about 1500 lU / mL and at or about 5000 lU / mL, between at or about 1500 lU / mL and at or about 4000 lU / mL, between at or about 1500 lU / mL and at or about 2000 lU / mL, between at or about 2000 lU / mL and at or about 5000 lU / mL, such as between at or about 2000 lU / mL and at or about 4000 lU / mL, or between at or about 4000 lU / mL and at or about 5000 lU / mL. In some embodiments, the recombinant IL-21 is added to the cell culture media at a concentration of at or about 500 lU / mL, at or about 600 lU / mL, at or about 700 lU / mL, at or about 800 lU / mL, at or about 900 lU / mL, at or about 1000 lU / mL, at or about 1100 lU / mL, at or about 1200 lU / mL, at or about 1300 lU / mL, at or about 1400 lU / mL, at or about 1500 lU / mL, at or about 1600 lU / mL, at or about 1700 lU / mL, at or about 1800 lU / mL, at or about 1900 lU / mL or at or about 2000 lU / mL, or any concentration between any of the foregoing. In some embodiments, IL-21 is added to the culture medium at a concentration of at or about 1000 lU / mL.

[0457] In some embodiments, recombinant IL-21 and IL-2 are added to the culture medium. In some embodiments, recombinant IL-21 is added at a concentration of 500 lU / mL to 2000 lU / mL (e.g. at or about 1000 lU / mL) and recombinant IL-2 is added at a concentration of 200lU / mL to 5000 lU / mL (e.g. at or about 3000 lU / mL). In some embodiments, the expansion is carried out in the presence of recombinant IL-21 added at 1000 lU / mL and recombinant IL-2 added at 3000 lU / mL. In some embodiments, at least one other recombinant modulatory cytokine from IL-7, IL- 15, IL-23, IL-25, IL- 27 or IL-35 is added to the culture medium.

[0458] In one aspect of provided methods, the tumor reactive T cells are directly sorted after tumor digest before being genetically modified, and only a single expansion step is carried out after the genetic modification, in which the population of expanded T cells is harvested as a therapeutic TIL composition. In such an example, tumor fragments are digested into a single cell suspension and provided as an input sample for sorting / selection for the tumor-reactive T cells thereof. Then, the selected cells are genetically modified using any of the techniques described in Section LD before the cells are expanded and harvested as a therapeutic TIL composition. In some embodiments, the expansion is carried out for a period of time to achieve a therapeutic dose. In some embodiments, the expansion is carried to achieve a fold expansion of the cells of from at or about 200-fold to at or about 3000-fold. In some embodiments, the expansion is carried out to achieve a therapeutic dose of at or about or greater than at or about 500 million total cells. In some embodiments, the expansion is carried out for 1-28 days, such as for at or about 7 to 28 days, 7 to 21 days, 7 to 14 day, such as at or about 7 days, 8 days, 9 days, 10 days, 11 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days or 28 days.

[0459] In some embodiments, provided herein is a method for manufacturing tumor-reactive T cells comprising (a) selecting cells surface positive for PD-1 and / orCD39 from an input sample comprising T cells from a subject that has a tumor to obtain selected cells from the sample; (b) performing an expansion by culture of the selected cells with one or more T-cell stimulating agent of lymphocytes under conditions to produce a population of expanded T cells; (c) genetically modifying the expanded cells; and (d) performing another expansion by culture of the genetically modified cells with one or more T-cell simulating agent of lymphocytes under conditions to produce a population of expanded T cells. In some embodiments, provided herein is a method for manufacturing tumor-reactive T cells comprising (a) selecting cells surface positive for PD-1 and / or CD39 from an input sample comprising T cells from a subject that has a tumor to obtain selected cells from the sample; (b) genetically modifying the expanded cells; and (c) performing an expansion by culture of the genetically modified cells with one or more T- cell simulating agent of lymphocytes under conditions to produce a population of expanded Tcells.. In some embodiments, the method includes harvesting the population of expanded T cells produced by the method for formulation as the therapeutic composition.

[0460] In some embodiments, provided herein is a method for manufacturing tumor-reactive T cells comprising (a) selecting cells surface positive for PD-1 and CD39 from an input sample comprising T cells from a subject that has a tumor to obtain selected cells from the sample; and (b) performing an expansion by culture of the selected cells with one or more T-cell stimulating agent of lymphocytes under conditions to produce a population of expanded T cells. In some embodiments, the method includes harvesting the population of expanded T cells produced by the method for formulation as the therapeutic composition.

[0461] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells, contains recombinant IL-2. In some embodiments, one or more other stimulating agent can be included such as one or more other recombinant cytokine from IL-7, IL-15, IL-21, IL-25, and / or IL-23, or an anti-CD3 antibody (e.g. OKT-3). In some cases in which an anti-CD3 antibody (e.g. OKT-3) the T cell stimulating agent(s) also can include a costimulating agent, such as provided by antigen-presenting feeder cells, such as PBMCs, or a soluble anti-CD28 antibody.

[0462] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL-2 and an anti-CD3 antibody.

[0463] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL-2, an anti-CD3 antibody, e.g. OKT-3, and antigen-presenting feeder cells, such as PBMCs.

[0464] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL- 15, IL-21 and an anti-CD3 antibody.

[0465] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL-15, IL-21, an anti-CD3 antibody, e.g. OKT-3, and antigen-presenting feeder cells, such as PBMCs.

[0466] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL- 15, IL-7 and an anti-CD3 antibody.

[0467] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL-15, IL-7, an anti-CD3 antibody, e.g. OKT-3, and antigen-presenting feeder cells, such as PBMCs.

[0468] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL-2, an anti-CD3 antibody, e.g. OKT-3, and an anti-CD28 antibody. In some embodiments, the anti-CD3 antibody and / or anti-CD28 antibody are soluble. In some embodiments, one or both of the anti-CD3 antibody and anti- CD28 antibody are bound to a solid surface, such as a bead (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0469] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL-2 an anti-CD3 antibody, e.g. OKT-3, and antigen-presenting feeder cells, such as PBMCs.

[0470] In particular embodiments, the incubation or culture of T cells also is carried out with nutrient containing media so that the cells can survive outside of the body. In embodiments of the provided methods, one or more of the steps can be carried out in serum-containing media, such as media containing human AB serum. The culture media containing the T cell stimulatory agent(s) can be a serum-free media.

[0471] In one embodiment, the serum free medium is OpTmizer CTS (LifeTech), Immunocult XF (Stemcell technologies), CellGro (CellGenix), TexMacs (Miltenyi), Stemline (Sigma), Xvivol5 (Lonza), PrimeXV (Irvine Scientific), or Stem XVivo (RandD systems). The serum-free medium can be supplemented with a serum substitute such as ICSR (immune cell serum replacement) from LifeTech. The level of serum substitute (e.g., ICSR) can be, e.g., up to 5%, e.g., about 1%, 2%, 3%, 4%, or 5%. In some embodiments, the serum-free media contains 0.5 mM to 5 mM of a dipeptide form of L-glutamine, such L-alanyl-L- glutamine (Glutamax™). In some embodiments, the concentration of the dipeptide form of L-glutamine, such as L-alanyl- L-glutamine, is from or from about 0.5 mM to 5 mM, 0.5 mM to 4 mM, 0.5 mM to 3 mM, 0.5 mM to 2 mM, 0.5 mM to 1 mM, 1 mM to 5 mM, 1 mM to 4 mM, 1 mM to 3 mM, 1 mM to 2 mM, 2 mM to 5 mM, 2 mM to 4 mM, 2 mM to 3 mM, 3 mM to 5 mM, 3 mM to 4 mM or 4 mM to 5 mM, each inclusive. In some embodiments, the concentration of the dipeptide form of L- glutamine, such as L-alanyl-L-glutamine, is or is about 2 mM.

[0472] In some embodiments, the cells are cultured at about 37 °C with about 5% CO2.

[0473] In some embodiments, the incubation with the T cell stimulatory agent(s) is carried out for at or about 1 day, such as generally at or about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or any range of time between any of the foregoing. In some embodiments, the incubation with the T cell stimulatory agent(s) is carriedout for 7 to 21 days, such as 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days, or any value between any of the foregoing. In some embodiments, the incubation is carried out for 7-14 days. In some embodiments, the incubation is carried out for 7-10 days. In some embodiments, the incubation is for at or about 7 days. In some embodiments, the incubation is for at or about 8 days. In some embodiments, the incubation is for at or about 9 days. In some embodiments, the incubation is for at or about 10 days.

[0474] In some embodiments, the incubation with the T cell stimulatory agent(s) is a minimal expansion such that it does not result in downregulation of the T cell activation marker (e.g. PD-1 and / or CD39). For instance, the incubation with the T cell stimulatory agent(s) in the initial expansion is a short culture so that the markers CD39 and / or PD1 are still present during the sorting step and the cells have not downregulated those markers.

[0475] In some embodiments, the incubation with the T cell stimulatory agent(s), such as for the initial expansion of T cells in the input sample, is carried out for at or about 1 days. In some embodiments, the incubation with the T cell stimulatory agent(s), such as for the initial expansion of T cells in the input sample, is carried out for at or about 2 days. In some embodiments, the incubation with the T cell stimulatory agent(s), such as for the initial expansion of T cells in the input sample, is carried out for at or about 3 days. In some embodiments, the incubation with the T cell stimulatory agent(s), such as for the initial expansion of T cells in the input sample, is carried out for at or about 4 days. In some embodiments, the incubation with the T cell stimulatory agent(s), such as for the initial expansion of T cells in the input sample, is carried out for at or about 5 days. In some embodiments, the incubation with the T cell stimulatory agent(s), such as for the initial expansion of T cells in the input sample, is carried out for at or about 6 days. In some embodiments, the incubation with the T cell stimulatory agent(s), such as for the initial expansion of T cells in the input sample, is carried out for at or about 7 days.

[0476] The incubation, such as for initial expansion of T cells in the input sample, can be carried out under GMP conditions. In some embodiments, the incubation is in a closed system, which in some aspects may be a closed automated system. In some embodiments, the culture media containing the T cell stimulatory agent(s) can be a serum-free media. In some embodiments, the incubation is carried out in a closed automated system and with serum-free media.I ll

[0477] In some embodiments, the initial expansion of cells under the one or more stimulatory conditions is in a culture vessel suitable for cell expansion. In some embodiments, the culture vessel is a gas permeable culture vessel, such as a G-Rex system (e.g. G-Rex 10, G- Rex 10M, G-Rex 100 M / 100M-CS or G-Rex 500 M / 500M-CS). In some embodiments the culture vessel is a microplate, flask, bar or other culture vessel suitable for expansion of cells in a closed system. In some embodiments, expansion can be carried out in a bioreactor. In some embodiments, the initial expansion can be carried out using a cell expansion system by transfer of the cells to gas permeable bags, such as in connection with a bioreactor (e.g. Xuri Cell Expansion System W25 (GE Healthcare)). In an embodiment, the cell expansion system includes a culture vessel, such as a bag, e.g. gas permeable cell bag, with a volume that is about 50 mL, about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L, or any value between any of the foregoing. In some embodiments, the process is automated or semi-automated. Examples of suitable bioreactors for the automated perfusion expansion include, but are not limited to, GE Xuri W25, GE Xuri W5, Sartorius BioSTAT RM 20 | 50, Finesse SmartRocker Bioreactor Systems, and Pall XRS Bioreactor Systems, or Miltenyi Prodigy. In some aspects, the expansion culture is carried out under static conditions. In some embodiments, the expansion culture is carried out under rocking conditions. The medium can be added in bolus or can be added on a perfusion schedule. In some embodiments, the bioreactor maintains the temperature at or near 37°C and CO2 levels at or near 5% with a steady air flow at, at about, or at least 0.01 L / min, 0.05 L / min, 0.1 L / min, 0.2 L / min, 0.3 L / min, 0.4 L / min, 0.5 L / min, 1.0 L / min, 1.5 L / min, or 2.0 L / min or greater than 2.0 L / min. In certain embodiments, at least a portion of the culturing is performed with perfusion, such as with a rate of 290 ml / day, 580 ml / day, and / or 1160 ml / day.

[0478] In some embodiments, the cells are seeded in an appropriate culture vessel (e.g. gas permeable bag) at a density of from 0.5 x 106cells / mL to 1.5 x 106cells / mL. In some embodiments, the density is at or about 0.5 x 106cells / mL, 0.75 x 106cells / mL, 1 x 106cells / mL, 1.25 x 106cells / mL or 1.5 x 106cells / mL, or any value between any of the foregoing.

[0479] In some aspects, cells are expanded in an automated closed expansion system that is perfusion enabled. Perfusions can continuously add media to the cells to ensure an optimal growth rate is achieved.

[0480] The expansion methods can be carried out under GMP conditions, including in a closed automated system and using serum free medium. In some embodiments, any one or more of the steps of the method can be carried out in a closed system or under GMP conditions. In certain embodiments, all process operations are performed in a GMP suite. In some embodiments, a closed system is used for carrying out one or more of the other processing steps of a method for manufacturing, generating or producing a cell therapy. In some embodiments, one or more or all of the processing steps, e.g., isolation, selection and / or enrichment, processing, culturing steps including incubation in connection with expansion of the cells, and formulation steps is carried out using a system, device, or apparatus in an integrated or self- contained system, and / or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and / or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and / or adjust various aspects of the processing, isolation, modifying, and formulation steps.

[0481] In some embodiments, the stimulated cells are collected and are cryofrozen. In some embodiments, for cryopreservation, the stimulated cells are formulated as a composition with a cryoprotectant. In some embodiments, the cryoprotectant is or comprises DMSO and / or glycerol. In some embodiments, compositions formulated for cryopreservation can be stored at low temperatures, such as ultra low temperatures, for example, storage with temperature ranges from -40 °C to -150 °C, such as or about 80 °C ± 6.0 ° C.

[0482] In some embodiments, the cryopreserved cells are prepared for subsequent steps by thawing.IL COMPOSITIONS AND PHARMACEUTICAL FORMULATIONS

[0483] Provided herein are compositions comprising a population of genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), wherein at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the total cells or total T cells in the composition comprise a genetic disruption at a target site of a target gene as described herein. In some embodiments, the target gene is SOCS1, GISH. PDCD1 (PD-1), TIG I T. RC3H1 (Roquin), CMIP (gene encoding C-Maf Inducing Protein), CD39, LAG3, TIM3, MAPK14, or RASA2 (gene encoding RAS p21 protein activator 2). In some embodiments, the genetic disruption is at a target site in any of such genes, such as any of the target sites as described herein.

[0484] In some embodiments, provided herein are compositions comprising a population of genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), wherein at least at orabout 70%, 75%, 80%, 85%, 90%, or 95% of the total cells or total T cells in the composition comprise a genetic disruption at a target site in a SOCS1 target gene.

[0485] In some embodiments, provided herein is a composition comprising a population of genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), wherein at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the total cells or total T cells in the composition comprise a genetic disruption at a target site in a C1SH target gene.

[0486] In some embodiments, provided herein is a composition comprising a population of genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), wherein at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the total cells or total T cells in the composition comprise a genetic disruption in a target site in a PDCD1 (PD-1) target gene.

[0487] In some of any the provided embodiments, the genetic disruption is an indel. In some embodiments, the genetic disruption inactivates all alleles of the target gene. In some embodiments, the genetic disruption knock out expression of the target gene. In some embodiments, the expression is genomic expression or is protein expression on the cell surface as determined by flow cytometry.

[0488] Among any of the provided TIL compositions are those in which the tumor reactive T cells are enriched for tumor reactive T cells compared to input sample from a tumor. In some embodiments, the TIL compositions contain primary T cells from a tumor from a subject that have been selected based on surface expression of PD-1 and CD39 and expanded ex vivo. In some embodiments, provided TIL compositions containing selected and expanded T cells can be produced by the provided ex vivo methods for producing TIL compositions. In some embodiments, provided TIL compositions contain primary T cells from a tumor from a subject that have been genetically modified.

[0489] In some embodiments, the provided TIL composition is a multiclonal population that exhibits TCR diversity and enrichment of T cell receptors (TCRs) reactive to tumor antigens. In some embodiments, the provided TIL composition is an oligoclonal population that exhibits TCR diversity and enrichment of different TCR clonotypes. In some embodiments, the TIL composition contains up to 40 different TCR clonotypes (e.g. Top40 clones) that make up at least 40% of the TCR frequency in the population. In some embodiments, the TIL composition contains up to 40 different TCR clonotypes that make up at least 50% of the TCR frequency in the population. In some embodiments, the TIL composition contains up to 40 different TCR clonotypes that make up at least 60% of the TCR frequency in the population. In someembodiments, the TIL composition contains up to 40 different TCR clonotypes that make up at least 70% of the TCR frequency in the population. In some of any of the above embodiments, the number of TCR clonotypes making up the percentage is 10 to 40 different TCR clonotypes. In some of any of the above embodiments, the number of TCR clonotypes making up the percentage is 20 to 40 different TCR clonotypes. In some of any of the above embodiments, the number of TCR clonotypes making up the percentage is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 different TCR clonotypes.

[0490] In some embodiments, the TIL composition contains 20 to 40 different TCR clonotypes that make up at least 40% of the TCR frequency in the population. In some embodiments, the TIL composition contains 20 to 40 different TCR clonotypes that make up at least 50% of the TCR frequency in the population. In some embodiments, the TIL composition contains 20 to 40 different TCR clonotypes that make up at least 60% of the TCR frequency in the population. In some embodiments, the TIL composition contains 20 to 40 different TCR clonotypes that make up at least 70% of the TCR frequency in the population.

[0491] In some embodiments, the top40 TCR clonotypes make up at least 75% of the TCR frequency in the population. In some embodiments, the top40 TCR clonotypes make up at least 80% of the TCR frequency in the population. In some embodiments, the top40 TCR clonotypes make up at least 85% of the TCR frequency in the population. In some embodiments, the top40 TCR clonotypes make up at least 90% of the TCR frequency in the population.

[0492] In some embodiments, the neoantigen reactivity of the TCR clonotypes is reactivity to at least one CD4 antigen and at least one CD8 antigen. In some embodiments, the neoantigen reactivity of the TCR clonotypes is for at least 2 peptide antigens in which as least one peptide antigen is a CD4 antigen and at least one peptide antigen is a CD8 antigen.

[0493] Various methods for assessing the TCR repertoire for clonotype identification and TCR repertoire analysis are known (see e.g. Rosati et al. (2017) BMC Biotechnology, 17:61; Friedensohn et al. (2016) Trends in Biotechnology, 35:203-214). In some aspects, the methods involve high-throughput or next-generation sequencing methods. In some embodiments, the frequency and variety of different clones present in the population or composition can be determined. In some embodiments, the compositions can be assessed the clonality, clonal diversity or clonal heterogeneity of the cells in the population of the composition of cells, for example, based on the determined frequency and / or variety of clonotypes present in thepopulation or composition. In some embodiments, single-cell sequencing methods are carried out to identify a clonotype on a particular cell. In certain aspects, paired aP TCR sequencing methods are used (see e.g. W02017053902A1). In some embodiments, sequencing methods are carried out on DNA, such as genomic DNA or complementary DNA. In some embodiments, sequencing methods are carried out on RNA. In some embodiments, high-throughput or nextgeneration sequencing of TCR sequences or by sequencing the whole genome or transcriptome (e.g., RNAseq). In some aspects, the methods used are RNAseq-based methods.

[0494] In some embodiments, T cell clonotype assessment and clonality and diversity in various T cell populations or compositions or samples containing T cells, are determined using high-throughput sequencing of all or a portion of the TCR genes or based on sequences obtained from high-throughput whole genome or transcriptome analysis, on the population or composition of cells, and / or in a single cell. In some embodiments, the provided methods can include various features of the methods as described in WO2016 / 044227, WO2016 / 176322, W02012 / 048340, WO2012 / 048341, WO2014 / 144495, W02017 / 053902, W02017 / 053903 or W02017 / 053905, each incorporated by reference in their entirety.

[0495] The clonotypes of a cell or the clonotypes present in a population or composition of cells, in some examples, may be determined by TCR sequencing. In some embodiments, sequencing methods that can be employed include high-throughput or next-generation sequencing as is known in the art. In some aspects, next-generation sequencing methods can be employed, using genomic DNA or cDNA from T cells, to assess the TCR repertoire, including sequences encoding the complementarity-determining region 3 (CDR3). In some embodiments, whole transcriptome sequencing by RNAseq can be employed. In some aspects, the TCR repertoire information, e.g., TCR sequences and relative frequency, can be constructed or extracted from whole transcriptome sequencing (e.g., by RNAseq). For example, in some aspects, computational methods such as MIXCR (Such as those described in Bolotin et al. Nature Methods 12 (2015) 380-381, Bolotin et al., Nature Biotechnology 35 (2017) 908-911) or IMREP (Mangul et al., bioRxiv (2017) 089235) can be utilized to determine the repertoire TCR sequences or a portion thereof (e.g., CDR3) from whole transcriptome RNAseq results. In some embodiments, single-cell sequencing methods can be used. In some embodiments, clonotypes can be assessed or determined by spectratype analysis (a measure of the TCR VP, Va, Vy, or V5 chain hypervariable region repertoire). Clonotypes can also be determined by generation and characterization of antigen- specific clones to an antigen of interest.

[0496] In some embodiments, T cell clonotype assessment are determined using high- throughput sequencing of all or a portion of the TCR genes or based on sequences obtained from high-throughput whole genome or transcriptome analysis, on the population or composition of cells, and / or in a single cell. In some embodiments, bulk sequencing of targeted sequences (e.g., TCR chains or portion thereof) or bulk whole genome or transcriptome sequencing (e.g., by RNAseq) can be used to determine the clonotypes present in the cells in the population or composition. In some aspects, T cell clonotype assessment can involve sequencing of a portion of the variable region of one or more native TCR chains, such as the complementaritydetermining region 3 (CDR3). In some aspects, single cell sequencing can be employed. In some embodiments, the provided methods can include various features of the methods as described in WO2016 / 044227, WO2016 / 176322, W02012 / 048340, WO2012 / 048341, WO20 14 / 144495, W02017 / 053902, W02017 / 053903 or W02017 / 053905, each incorporated by reference in their entirety. In some embodiments, for target TCR molecules, the genes encoding chains of a TCR can be obtained from genomic DNA or mRNA of immune cells or T cells.

[0497] In some embodiments, the composition exhibits clonal diversity, i.e. is multiclonal, such as is oligoclonal. In some cases, the clonal diversity is determined based on the relative frequency of the one or more clonotypes and / or one or more TCR sequences. In some embodiments, the determining the clonal diversity is represented as clonality, Shannon-adjusted clonality or top 25 clonality of each of the plurality of samples. In some embodiments, the determining the clonal diversity is represented as Shannon-adjusted clonality in a composition.

[0498] In some embodiments, the cells of the provided TIL compositions exhibit one or more phenotypic or functional markers. In some cases, such cells include cells positive or negative for one or more phenotypic marker or functional feature or attribute.

[0499] As used herein, a statement that a cell or population of cells is “positive” for a particular marker, function or attribute refers to the detectable presence on or in the cell of a particular marker, such as a surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and / or at a level substantially similar to that for cell known to be positive for themarker, and / or at a level substantially higher than that for a cell known to be negative for the marker.

[0500] As used herein, a statement that a cell or population of cells is “negative” for a particular marker, function or attribute refers to the absence of substantial detectable presence on or in the cell of a particular marker, such as a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and / or at a level substantially lower than that for cell known to be positive for the marker, and / or at a level substantially similar as compared to that for a cell known to be negative for the marker.

[0501] Exemplary markers, functions and attributes of provided TIL compositions are described below. In some embodiments, the TIL composition is characterized by any one or more of such features, such as 2, 3, 4, 5 or more of such features. Lor instance, a provided TIL composition may be characterized by presence or absence of one or more T cell markers, effector memory phenotype markers, exhaustion markers, the ability to produce or secrete cytokines and / or the ability to produce or secrete a cytotoxic factor, such as described below. Any 2, 3, 4, 5 or more of any of such features may be present in a TIL composition as described.

[0502] In some embodiments, a provided TIL composition comprises CD3+ T cells as a percentage of total cells in the population that is greater than or greater than about 85%, such as greater than or greater than about 90%, such as greater than or greater than about 95%, greater than or greater than about 97% or greater than or greater than about 98%. In some embodiments, a provided TIL composition comprises CD3+ T cells as a percentage of total cells in the population that is greater than or greater than about 90%. In some embodiments, a provided TIL composition comprises CD3+ T cells as a percentage of total cells in the population that is greater than or greater than about 95%. In some embodiments, a provided TIL composition comprises CD3+ T cells as a percentage of total cells in the population that is greater than or greater than about 98%. In some embodiments, the composition contains CD4+ T cells and CD8+ T cells as a percentage of total cells in the population that is greater than or greater than about 85%, greater than or greater than about 90%, greater than or greater than about 95%, greater than or greater than about 97% or greater than or greater than about 98%.

[0503] In some embodiments, the composition contains a ratio of CD4+ T cells to CD8+ T cells that is between at or about ...

Claims

CLAIMSWHAT IS CLAIMED:

1. A method of generating genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), the method comprising:(a) selecting cells surface positive for one or more T cell activation markers from a tumor sample from a subject to produce a population of tumor-reactive TIL;(b) activating T cells of the population of tumor-reactive TIL by incubating the T cells with a T cell activation reagent that comprises an anti-CD3 antibody or antigen binding fragment thereof and an anti-CD28 antibody or antigen binding fragment thereof to generate an activated population of tumor-reactive TIL, wherein the activation is carried out for 1 to 6 days;(c) introducing a genetic disruption at a target site within a target gene in T cells of the activated population of tumor-reactive TIL by gene editing to produce a population of genetically modified tumor-reactive TIL;(d) performing an expansion by culturing the population of genetically modified tumor- reactive TIL with one or more T-cell stimulating agents to produce a population of genetically modified and expanded tumor-reactive TIL, wherein one of the one or more T-cell stimulating agents is an anti-CD3 antibody or antigen binding fragment thereof the expansion is carried out for 7 to 21 days; and(e) harvesting the population of genetically modified and expanded tumor-reactive TIL from step (d).

2. A method of generating genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), the method comprising:(a) selecting cells surface positive for one or more T cell activation markers from a tumor sample from a subject to produce a population of tumor-reactive TIL;(b) activating T cells of the population of tumor-reactive TIL by incubating the T cells with a T cell activation reagent that comprises an anti-CD3 antibody or antigen binding fragment thereof and an anti-4- IBB antibody or antigen binding fragment thereof to generate an activated population of tumor-reactive TIL, wherein the activation is carried out for 1 to 6 days;(c) introducing a genetic disruption at a target site within a target gene in T cells of the activated population of tumor-reactive TIL by gene editing to produce a population of genetically modified tumor-reactive TIL;(d) performing an expansion by culturing the population of genetically modified tumor- reactive TIL with one or more T-cell stimulating agent to produce a population of genetically modified and expanded tumor-reactive TIL, wherein one of the one or more T-cell stimulating agents is an anti-CD3 antibody or antigen binding fragment thereof the expansion is carried out for 7 to 21 days; and(e) harvesting the population of genetically modified and expanded tumor-reactive TIL from step (d).

3. A method of generating genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), the method comprising:(a) obtaining a population of tumor-reactive TIL from a tumor sample from a subject;(b) introducing a genetic disruption at a target site within a target gene selected from the group consisting of SOCS1, GISH. PDCD1 (PD-1), TIGIT, RC3HI (Roquin), CMIP (gene encoding C-Maf Inducing Protein), CD39, LAG3, TIM3, MAPK14, and RASA2 (gene encoding RAS p21 protein activator 2) in T cells of the population of tumor-reactive TIL by gene editing to produce a population of genetically modified tumor-reactive TIL;(c) performing an expansion by culturing the population of genetically modified tumor- reactive TIL with one or more T-cell stimulating agent to produce a population of genetically modified and expanded tumor-reactive TIL; and(d) harvesting the population of genetically modified and expanded tumor-reactive TIL from step (c).

4. The method of claim 3, wherein the population of tumor-reactive TIL are selected TIL in which cells from the tumor sample are selected for cells surface positive for one or more T cell activation marker.

5. The method of any of claims 1-2 and 4, wherein the one or more T cell activation markers is selected from the group consisting of PD-1, CD39, TIGIT, CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (0X40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4),TIM-3 and LAG-3.

6. The method of any of claims 1, 2, 4 and 5, wherein the one or more T cell activation markers comprise CD39 and at least one other marker selected from the group consisting of PD-1, TIGIT, CD107a, CD103, CD137 (4- IBB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (0X40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM-3 and LAG-3.

7. The method of any of claims 1, 2, 4-6, wherein the one or more T cell activation markers are PD-1 and / or CD39.

8. The method of any of claims 1, 24-7, wherein the one or more T cell activation markers are PD-1 and CD39.

9. The method of any of claims 1, 2 and 4-8, wherein the population of tumor- reactive TIL are T cells from the tumor sample further selected for cells surface positive for CD45, CD4 and / or CD 8, optionally CD45 and CD4 and / or CD 8.

10. The method of any of claims 1-9, wherein the population of tumor-reactive TIL is an oligoclonal population that is enriched for T cells expressing a T cell receptor (TCR) reactive to tumor antigen.

11. A method of generating genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), the method comprising:(a) selecting cells surface positive for one or more T cell activation markers from a tumor sample from a subject to produce a population of tumor-reactive TIL; and(b) introducing a genetic disruption at a target site within a target gene selected from the group consisting of SOCS1, GISH. PDCD1 (PD-1), TIGIT, RC3H1 (Roquin), CMIP (gene encoding C-Maf Inducing Protein), CD39, LAG3, TIM3, MAPK14, and RASA2 (gene encoding RAS p21 protein activator 2) in T cells of the population of tumor-reactive TIL by gene editing to produce a population of genetically modified tumor-reactive TIL;(c) performing an expansion by culturing the population of genetically modified tumor- reactive TIL with one or more T-cell stimulating agent to produce a population of genetically modified and expanded tumor-reactive TIL; and(d) harvesting the population of genetically modified and expanded tumor-reactive TIL from step (c).

12. The method of claim 11, wherein the one or more T cell activation markers are selected from the group consisting of PD-1, CD39, TIGIT, CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (0X40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM- 3 and LAG- 3.

13. The method of claim 11 or claim 12, wherein the one or more T cell activation markers comprise CD39 and at least one other marker selected from the group consisting of PD- 1, TIGIT, CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (0X40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM-3 and LAG-3.

14. The method of any of claims 11-13, wherein the one or more T cell activation markers are PD-1 and / or CD39.

15. The method of any of claims 11-14, wherein the one or more T cell activation markers are PD-1 and CD39.

16. The method of any of claims 1, 2, 4-10, 11-15, wherein in step (a) the cells are further selected for cells positive for the surface marker CD45.

17. The method of any of claims 1, 2, 4-10, 11-16, wherein in step (a) the cells are further selected for cells positive for the surface marker CD3.

18. The method of any of claims 1, 2, 4-10, 11-17, wherein in step (a) the cells are further selected for cells positive for the surface markers CD4 and / or CD8.

19. The method of any of claims 1, 2, 4-10, 11-18, wherein in step (a) the cells are further selected for cells positive for surface markers CD4 and CD8.

20. The method of any of claims 1, 2 and 4-19, wherein the cells are selected for the marker by contacting cells from the tumor sample with a binding agent that binds to the marker and isolating cells positive for the marker.

21. A method of generating genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), the method comprising:(a) contacting cells from a tumor sample from a subject with a binding agent(s) that detects one or more T cell activation markers;(b) selecting cells from step (a) positive for the one or more T cell activation markers to produce a population of tumor-reactive TIL;(c) introducing a genetic disruption at a target site within a target gene selected from the group consisting of SOCS1, GISH. PDCD1 (PD-1), TIGIT, RC3HI (Roquin), CMIP (gene encoding C-Maf Inducing Protein), CD39, LAG3, TIM3, MAPK14, and RASA2 (gene encoding RAS p21 protein activator 2) in T cells of the population of tumor-reactive TIL by gene editing to produce a population of genetically modified tumor-reactive TIL;(d) performing an expansion by culturing the population of genetically modified tumor- reactive TIL with one or more T-cell stimulating agent to produce a population of genetically modified and expanded tumor-reactive TIL; and(e) harvesting the population of genetically modified and expanded tumor-reactive TIL from step (d).

22. The method of claim 21, wherein the one or more T cell activation markers are selected from the group consisting of PD-1, CD39, TIGIT, CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (0X40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM- 3 and LAG- 3.

23. The method of claim 21 or 22, wherein the one or more T cell activation markers comprise CD39 and at least one other marker selected from the group consisting of PD-1, TIGIT, CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (0X40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM-3 and LAG-3.

24. The method of any of claims 21-23, wherein the one or more T cell activation markers are PD-1 and / or CD39.

25. The method of any of claims 21-24, wherein the one or more T cell activation markers are PD-1 and CD39.

26. The method of claim 21-25, wherein in step (a) the cells from the tumor sample are further contacted with a binding agent that detects CD45 and in step (b) selecting cells surface positive for CD45.

27. The method of any of claims 21-26, wherein in step (a) the cells are further contacted with a binding agent that detects CD3 and in step (b) selecting for cells surface positive for CD3.

28. The method of any of claims 21-27, wherein in step (a) the cells are further contacted with binding agent(s) that detect CD4 and / or CD8 and in step (b) selecting for cells surface positive for CD4 and / or CD8.

29. The method of any of claims 21-28, wherein in step (a) the cells are further contacted with binding agent(s) that detects CD4 and CD8 and in step (b) selecting for cells surface positive for CD4 and CD8.

30. The method of any of claims 3-29, wherein prior to introducing the genetic disruption, the method comprises incubating the population of tumor-reactive TIL with a T cell activation reagent to produce a population of activated TIL.

31. The method of claim 30, wherein the T cell activation reagent comprises an anti-CD3 antibody or antigen binding fragment thereof.

32. The method of claim 31, wherein the anti-CD3 antibody or antigen binding fragment thereof is a primary T cell activation agonist, and the T cell activation reagent further comprises a costimulatory agonist.

33. The method of claim 32, wherein the costimulatory agonist is an anti-CD28 antibody or antigen binding fragment thereof.

34. The method of claim 32, wherein the costimulatory agonist is an anti-4- IBB antibody or antigen binding fragment thereof.

35. The method of any of claims 30-33, wherein the T cell activation reagent comprises an anti-CD3 antibody or antigen binding fragment thereof and an anti-CD28 antibody or antigen binding fragment thereof.

36. The method of any of claims 30-33, 34, and 35, wherein the T cell activation reagent comprises an anti-CD3 antibody or antigen binding fragment thereof and an anti-4- IBB antibody or antigen binding fragment thereof.

37. The method of any of claims 1, 2 and 30-36, wherein the incubating of the population of tumor-reactive TIL with the T cell activation reagent takes place for about 1 days to 5 days.

38. The method of any of claims 1, 2 and 30-37, wherein the incubating of the population of tumor-reactive TIL with the T cell activation reagent takes place for about 2 days to 4 days.

39. The method of any of claims 1, 2, 5-10, 16-20 and 30-38, wherein the incubating of the population of tumor-reactive TIL with the T cell activation reagent takes place for about 3 days.

40. The method of any of claims 1, 2, 5-10, 16-20 and 30-39, wherein the incubating of the population of tumor-reactive TIL with the T cell activation reagent is performed without feeder cells.

41. The method of any of claims 3-29, wherein prior to introducing the genetic disruption, the method comprises incubating the population of tumor-reactive TIL with a first T cell activation reagent to produce a first activated population of TILs and incubating the first activated population of TIL with a second T cell activation reagent for producing a second activated population of TIL.

42. The method of claim 41, wherein the first T cell activation reagent comprises an anti-CD3 antibody or antigen binding fragment thereof and an anti-4- IBB antibody or antigen binding fragment thereof and / or a 4- IBB agonist.

43. The method of claim 41 or claim 42, wherein the second T cell activation reagent comprises an anti-CD3 antibody or antigen binding fragment thereof.

44. The method of claim 43, wherein the second T cell activation reagent further comprises an anti-CD28 antibody.

45. The method of claim 43, wherein the second T cell activation reagent further comprises an anti-4- IBB antibody and / or 4- IBB agonist.

46. A method of generating genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), the method comprising:(a) selecting cells surface positive for one or more T cell activation markers from a tumor sample from a subject to produce a population of tumor-reactive TIL;(b) incubating T cells of the population of tumor-reactive TIL by incubating the T cells with a first T cell activation reagent that comprises an anti-CD3 antibody or antigen binding fragment thereof and an anti-4- IBB antibody or antigen binding fragment thereof to generate a first activated population of TIL;(c) incubating the T cells of the first activated population of TIL with a second T cell activation reagent that comprises an anti-CD3 antibody or antigen binding fragment thereof and an anti-CD28 antibody or antigen binding fragment thereof to generate a second activated population of TIL;(d) introducing a genetic disruption at a target site within a target gene in T cells of the second activated population of TIL by gene editing to produce a population of genetically modified tumor-reactive TIL;(e) performing an expansion by culturing the population of genetically modified tumor- reactive TIL with one or more T-cell stimulating agent to produce a population of genetically modified and expanded tumor-reactive TIL, wherein one of the one or more T-cell stimulating agents is an anti-CD3 antibody or antigen binding fragment thereof the expansion is carried out for 7 to 21 days; and(f) harvesting the population of genetically modified and expanded tumor-reactive TIL from step (d).

47. The method of any of claims 41-46, wherein the incubating of the population of tumor-reactive TIL with the first T cell activation reagent takes place for about 2 days to 14 days.

48. The method of any of claims 41-47, wherein the incubating of the population of tumor-reactive TIL with the first T cell activation reagent takes place for about 10 days to 12 days.

49. The method of any of claims 41-48, wherein the incubating of the population of tumor-reactive TIL with the first T cell activation reagent takes place for about 11 days.

50. The method of any of claims 41-47, wherein the incubating of the population of tumor-reactive TIL with the first T cell activation reagent takes place for about 6 days to 8 days.

51. The method of any of claims 41-47 and 50, wherein the incubating of the population of tumor-reactive TIL with the first T cell activation reagent takes place for about 7 days.

52. The method of any of claims 41-51, wherein incubating of the first activated population of TIL with the second T cell activation reagent takes place for about 2 days to 4 days.

53. The method of any of claims 41-52, wherein incubating the first activated population of TIL with the second T cell activation reagent takes place for about 3 days.

54. The method of any of claims 41-53, wherein incubating the population of tumor- reactive TIL with the first T cell activation reagent and / or incubating the first activated population of TIL with the second T cell activation reagent is performed without feeder cells.

55. The method of any of claims 1-40, wherein incubating the population of tumor- reactive TIL with the T cell activation reagent is carried out in the presence of recombinant IL-2.

56. The method of any of claims 41-53, wherein the incubating the population of tumor-reactive TIL with the first T cell activation reagent and / or the incubating the first activated population of TIL with the second T cell activation reagent is carried out in the presence of recombinant IL-2.

57. The method of claim 55 or claim 56, wherein the recombinant IL-2 is at a concentration of between at or about 1000 lU / mL to 6,000 lU / mL, optionally at a concentration of about 3,000 lU / mL.

58. The method of any of claims 1, 2, 5-10, 16-20, 30-40, 55 and 57, wherein the activated population of tumor-reactive TIL is introduced with the genetic disruption within about 3 days of incubating with the first T cell activation reagent.

59. The method of any of claims 41-54, 56 and 57, wherein the second activated population of TIL is introduced with the genetic disruption within about 3 days of incubating with the second T cell activation reagent.

60. The method of any of claims 1-59, wherein the genetic disruption is introduced using a gene editing agent comprising a CRISPR-Cas combination comprising a guide RNA (gRNA) that binds to the target site and a Cas nuclease.

61. The method of claim 60, wherein the Cas nuclease is a Cas9.

62. The method of claim 61, wherein the Cas9 is a streptococcus pyogenes Cas9 (spCas9).

63. The method of claim 60, wherein the Cas nuclease is Mad7.

64. The method of any of claims 60-63, wherein the gene editing agent is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease.

65. The method of any of claims 60-63, wherein the gene editing agent is a delivery vector comprising a polynucleotide encoding the Cas9 nuclease and comprising the gRNA.

66. The method of claim 65, wherein the delivery vector is a lipid nanoparticle or is a viral vector.

67. The method of any of claims 1-66, wherein the gene editing agent is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease.

68. The method of claim 67, wherein the RNP complex is delivered to the T cells of the population of tumor-reactive TIL using electroporation.

69. The method of claim 67, wherein the RNP complex is delivered to the T cells of the population of tumor-reactive TIL using an amphiphilic peptide reagent.

70. The method of claim 69, wherein the RNP complex is mixed with the amphiphilic peptide reagent prior to the introducing step.

71. The method of claim 69 or 70, wherein the RNP complex is delivered using peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC).

72. The method of any of claims 1-71, wherein the target gene is SOCSL C1SH or PD-1.

73. The method of any of claims 1-72, wherein the genetic disruption is at a target site within the SOCS1 locus.

74. The method of claim 73, wherein the genetic disruption is at a target site within exon 2.

75. The method of claim 73 or claim 74, wherein the genetic disruption is in the SH2 domain.

76. The method of any of claims 1-75, wherein the genetic disruption is in a target site within genomic coordinates chl6:l l, 255, 013-11, 255, 255.

77. The method of any of claims 1-76, wherein the target site comprises the sequence set forth in any one of SEQ ID NOS: 13-17, optionally wherein the target site is set forth in any one of SEQ ID Nos: 13-17.

78. The method of any of claims 1-77, wherein the target site comprises the sequence set forth in SEQ ID NO: 13, optionally wherein the target site is set forth in SEQ ID NO: 13.

79. The method of any of claims 1-77, wherein the target site comprises the sequence set forth in SEQ ID NO: 14, optionally wherein the target site is set forth in SEQ ID NO: 14.

80. The method of any of claims 1-79, wherein the gRNA comprises a spacer sequence set forth in any one of SEQ ID NOS: 35-39.

81. The method of any of claims 1-78 and 80, wherein the gRNA comprises a spacer sequence set forth in SEQ ID NO: 35.

82. The method of any of claims 1-77, 79 and 80, wherein the gRNA comprises a spacer sequence set forth in SEQ ID NO: 36.

83. The method of any of claims 1-75, wherein the target site comprises the sequence set forth in any one of SEQ ID NOS: 21-22, optionally wherein the target site is set forth in SEQ ID NO: 21 or SEQ ID NO:22.

84. The method of any of claims 1-75 and 83, wherein the gRNA comprises a spacer sequence set forth in SEQ ID NO: 43 or SEQ ID NO: 44.

85. The method of claim 73 or claim 74, wherein the genetic disruption is in the KIR domain.

86. The method of any of claims 1-74 and 85, wherein the genetic disruption is in a target site within genomic coordinates chl6:l l, 255, 287-11, 255, 329.

87. The method of any of claims 1-74, 85 and 86, wherein the target site comprises the sequence set forth in any one of SEQ ID NOS: 18-20, optionally wherein the target site is set forth in any one of SEQ ID Nos: 18-20.

88. The method of any of claims 1-74, and 85-87, wherein the target site comprises the sequence set forth in SEQ ID NO: 20, optionally wherein the target site is set forth in SEQ ID NO: 20.

89. The method of any of claims 1-74, and 85-88, wherein the gRNA comprises a spacer sequence set forth in any one of SEQ ID NOS: 40-42.

90. The method of any of claims 1-74, and 85-89, wherein the gRNA comprises a spacer sequence set forth in SEQ ID NO: 42.

91. The method of any of claims 1-73, wherein the genetic disruption is at a target site within the C1SH locus.

92. The method of claim 91, wherein the genetic disruption is at a target site within exon 3.

93. The method of any of claims 1-73, 91 and 92, wherein the genetic disruption is in a target site within genomic coordinates ch3: 50,607,687-50,608,132.

94. The method of any of claims 1-73, and 91-93, wherein the target site comprises the sequence set forth in any one of SEQ ID NOS: 1-7, optionally wherein the target site is set forth in any one of SEQ ID NOS: 1-7.

95. The method of any of claims 1-73, and 91-94, wherein the target site comprises the sequence set forth in any one of SEQ ID NOS: 1-5, optionally wherein the target site is set forth in any one of SEQ ID NOS: 1-5.

96. The method of claim 94 or claim 95, wherein the target site comprises the sequence set forth in SEQ ID NO: 5, optionally wherein the target site is set forth in SEQ ID NO:5.

97. The method of any of claims 1-73 and 91-96, wherein the gRNA comprises a spacer sequence set forth in any one of SEQ ID NOS: 23-29.

98. The method of any of claims 1-73 and 91-97, wherein the gRNA comprises a spacer sequence set forth in any one of SEQ ID NOS: 23-27.

99. The method of any of claims 1-73 and 91-98, wherein the gRNA comprises a spacer sequence set forth in SEQ ID NO: 27.

100. The method of any of claims 1-73, wherein the genetic disruption is at a target site within the PD-1 locus.

101. The method of claim 100, wherein the genetic disruption is at a target site within exon 2, 3 or 5.

102. The method of any of claims 1-73, 100 and 101, wherein the genetic disruption is in a target site within genomic coordinates ch2:241, 852, 672-241, 851, 173.

103. The method of any of claims 1-73 and 100-102, wherein the target site comprises the sequence set forth in any one of SEQ ID NOS: 8-12, optionally wherein the target site is set forth in any one of SEQ ID NOS: 8-12.

104. The method of claim 103, wherein the target site comprises the sequence set forth in SEQ ID NO: 10, optionally wherein the target site is set forth in SEQ ID NO: 10.

105. The method of any of claims 1-73 and 100-104, wherein the gRNA comprises a spacer sequence set forth in any one of SEQ ID NOS: 30-34.

106. The method of any of claims 1-73 and 100-105, wherein the gRNA comprises a spacer sequence set forth in SEQ ID NO:32.

107. The method of any of claims 1-106, wherein the genetic disruption comprises an indel in the target gene in cells of population of genetically modified cells.

108. The method of any of claims 1-107, wherein the genetic disruption inactivates all alleles of the target gene in cells of population of genetically modified cells.

109. The method of any of claims 1-108, wherein the genetic disruption is a knockout of the target gene in cells of population of genetically modified cells.

110. The method of any of claims 1-109, wherein the one or more T-cell stimulating agent of lymphocytes is an anti-CD3 agent (e.g. OKT3) and / or a recombinant cytokine selected from one or more of IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL- 27 and IL-35.

111. The method of any of claims 1-110, wherein the one or more T-cell stimulating agents of lymphocytes comprises one or more recombinant cytokines selected form IL-2, IL15, and IL-21.

112. The method of any of claims 1-111, wherein at least one of the one or more T- cell stimulating agent is recombinant IL-2.

113. The method of any of claims 110-112, wherein the concentration of recombinant IL-2 added to the culture for the expansion is from 100 lU / mL to 6000 lU / mL, such as from 300 lU / mL to 3000 lU / mL.

114. The method of claim 113, wherein the concentration of IL-2 added to the culture for the expansion is at or about 3000 lU / mL.

115. The method of any of claims 1-111, wherein the one or more T-cell stimulating agents does not include recombinant IL-2.

116. The method of any of claims 1-111, wherein the expansion is performed without the presence of recombinant IL-2.

117. The method of any of claims 1-116, wherein at least one of the T-cell stimulating agent is IL- 15 and / or IL-21.

118. The method of any of claims 1-117, wherein the expansion is performed in the presence of IL- 15 and IL-21.

119. The method of claim 117 or claim 118, wherein the concentration of IL- 15 added to the culture for expansion is from 250 lU / mL to 1500 lU / mL, such as from 250 lU / mL to 750 lU / mL.

120. The method of claim 88, wherein the concentration of IL- 15 added to the culture for the expansion is at or about 500 lU / mL.

121. The method of any of claims 117-120, wherein the concentration of IL-21 added to the culture for expansion is from 0.5 lU / mL to 10 lU / mL, such as from 1 lU / mL to 5 lU / mL.

122. The method of any of claims 117-121, wherein the concentration of IL-21 added to the culture for the expansion is at or about 1.15 lU / mL.

123. The method of any of claims 1-122, wherein at least one of the T-cell stimulating agent is an anti-CD3 antibody, optionally wherein the anti-CD3 antibody is OKT3.

124. The method of claim 123, wherein the anti-CD3 antibody is added to the culture at a concentration of from about 10 ng / mL to about 100 ng / mL, such as at a concentration of from about 20 ng / mL to about 50 ng / mL.

125. The method of claim 124, wherein the anti-CD3 antibody is added to the culture at a concentration of about 30 ng / mL.

126. The method of any of claims 110-125, wherein the one or more T-cell stimulating agent further comprises feeder cells that are non-dividing peripheral blood mononuclear cells (PBMC), optionally wherein the feeder cells are gamma irradiated.

127. The method of claim 126, wherein the feeder cells are present at a ratio of feeder cells to TIL cells of from about 50:1 to 500:1, optionally wherein the ratio of feeder cells to TIL cells is at or about 200: 1.

128. The method of any of claims 1-114 and 123-127, wherein the one or more T cell stimulating agent comprises about 3000 lU / mL IL-2, 30 ng / mL anti-CD3 antibody (OKT3) and gamma irradiated PBMC feeder cells at a ratio of about 200: 1 feeder cells to TIL.

129. The method of any of claims 1-128, wherein the performing the expansion to produce the expanded population of T cells is for 7 to 35 days.

130. The method of any of claims 1-129, wherein the performing the expansion to produce the expanded population of T cells is for 7 to 28 days, optionally 14 days to 28 days.

131. The method of any of claims 1-130, wherein the performing the expansion to produce the expanded population of T cells is for 7 to 21 days, optionally 7 to 14 days.

132. The method of any of claims 1-131, wherein the expansion is a second expansion and the method comprises performing a first expansion prior to introducing the genetic disruption at a target site in T cells of the population of tumor-reactive TIL, wherein the first expansion is by culturing the population of tumor-reactive TIL with one or more first T-cell stimulating agent.

133. The method of any of claims 1-131, wherein the expansion is a second expansion, and the method comprises performing a first expansion prior to the second expansion but subsequent to the introducing a genetic disruption, wherein the first expansion is by culture of the population of tumor-reactive TIL with one or more first T-cell stimulating agent.

134. The method of claim 132 or claim 133, wherein the one or more first T-cell stimulating agent of lymphocytes is an anti-CD3 agent (e.g. OKT3) and / or a recombinant cytokine selected from one or more of IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27 and IL-35.

135. The method of any of claims 132-134, wherein at least one of the one or more first T-cell stimulating agent is recombinant IL-2.

136. The method of claim 134 or claim 135, wherein the concentration of recombinantIL-2 added to the culture for the first expansion is from 100 lU / mL to 6000 lU / mL, such as from300 lU / mL to 3000 lU / mL.

137. The method of claim 136, wherein the concentration of IL-2 added to the culture for the first expansion is at or about 3000 lU / mL.

138. The method of any of claims 132-137, wherein at least one of the T-cell stimulating agent is IL- 15 and / or IL-21.

139. The method of claim 138, wherein the concentration of IL- 15 added to the culture for the first expansion is from 250 lU / mL to 1500 lU / mL, such as from 250 lU / mL to 750 lU / mL.

140. The method of claim 138 or claim 139, wherein the concentration of IL- 15 added to the culture for the first expansion is at or about 500 lU / mL.

141. The method of any of claims 138-140, wherein the concentration of IL-21 added to the culture for the first expansion is from 0.5 lU / mL to 10 lU / mL, such as from 1 lU / mL to 5 lU / mL.

142. The method of any of claims 138-141, wherein the concentration of IL-21 added to the culture for the first expansion is at or about 1.15 lU / mL.

143. The method of any of claims 132-142, wherein at least one of the first T-cell stimulating agent is an anti-CD3 antibody, optionally wherein the anti-CD3 antibody is OKT3.

144. The method of claim 143, wherein the anti-CD3 antibody is added to the culture at a concentration of from about 10 ng / mL to about 100 ng / mL, such as at a concentration of from about 20 ng / mL to about 50 ng / mL.

145. The method of claim 144, wherein the anti-CD3 antibody is added to the culture at a concentration of about 30 ng / mL.

146. The method of any of claims 134-145, wherein the one or more first T-cell stimulating agent further comprises feeder cells that are non-dividing peripheral blood mononuclear cells (PBMC), optionally wherein the feeder cells are gamma irradiated.

147. The method of claim 146, wherein the feeder cells are present at a ratio of feeder cells to TIL cells of from about 50:1 to 500:1, optionally wherein the ratio of feeder cells to TIL cells is at or about 200: 1.

148. The method of any of claims 132-147, wherein the one or more first T cell stimulating agents comprise about 3000 lU / mL IL-2, 30 ng / mL anti-CD3 antibody (OKT3) and gamma irradiated PBMC feeder cells at a ratio of about 200: 1 feeder cells to TIL.

149. The method of any of claims 132-148, wherein the performing the first expansion to produce the expanded population of T cells is for 7 to 35 days.

150. The method of any of claims 132-149, wherein the performing the expansion to produce the expanded population of T cells is for 7 to 28 days, optionally 14 days to 28 days.

151. The method of any of claims 132-150, wherein the performing the expansion to produce the expanded population of T cells is for 7 to 21 days, optionally 7 to 14 days.

152. The method of any of claims 1-151, wherein the input sample comprising T cells is derived from a resected tumor.

153. The method of claim 152, wherein the input sample comprising T cells is a single cell suspension processed by homogenization and / or enzymatic digestion of one or more tumor fragments from the resected tumor.

154. The method of claim 152 or claim 153, wherein the input sample comprising T cells is a single cell suspension processed by homogenization and enzymatic digestion of one or more tumor fragments from the resected tumor.

155. The method of claim 153 and claim 154, wherein the enzymatic digestion is by incubation with a collagenase, optionally collagenase IV or collagenase I / II.

156. The method of any of claims 1, 2 and 4-155, wherein the selecting for each of the one or more T cell activation markers is carried out simultaneously or sequentially in any order to obtain the population of tumor-reactive TIL.

157. The method of any of claims 1, 2 and 4-155, wherein the selecting is by flow cytometry.

158. The method of claim 157, wherein selecting the cells comprises sorting the cells by flow cytometry for cells positive for one or more fluorescence signal for detecting the one or more T cell activation markers, optionally at least 4 fluorescence signals, based on a fluorescence minus one (FMO) cocktail.

159. The method of claim 158, wherein the sorting is performed at a rate of 5,000 events per second to 10,000 events per second, optionally at about 6,000 events per second160. The method of any of claims 157-159, wherein selecting cells is performed using a microfluidics chip based cell sorting comprising one or more fluorescence detector for a fluorscenct signal for detecting the one or more T cell activation markers, optionally at least 4 fluorescence detectors.

161. The method of any of claims 1-160, wherein the performing the expansion is carried out in a closed system using a gas permeable membrane.

162. The method of any of claims 1-160, wherein the performing the expansion is carried out in a closed system using a bioreactor.

163. The method of any of claims 132-160, wherein the performing the first expansion is carried out in a closed system using a gas permeable culture vessel.

164. The method of any of claims 132-160, wherein the performing the first expansion is carried out in a closed system using a bioreactor.

165. The method of any of claims 132-164, wherein the performing the second expansion is performed in a gas permeable culture vessel.

166. The method of any of claims 132-164, wherein the performing the second expansion is performed using a bioreactor.

167. The method of any of claims 1-166, wherein the tumor is a tumor of an epithelial cancer.

168. The method of any of claims 1-167, wherein the tumor is a tumor of a melanoma, lung squamous, lung adenocarcinoma, bladder cancer, lung small cell cancer, esophageal cancer, colorectal cancer (CRC), cervical cancer, head and neck cancer, stomach cancer or uterine cancer.

169. The method of any of claims 1-168, wherein the tumor is a melanoma.

170. The method of any of claims 1-169, wherein the tumor is a colorectal cancer (CRC).

171. The method of any of claims 1-170, wherein the tumor is a tumor of a non- small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma cancer, endometrial cancer, optionally wherein the breast cancer is HR+ / Her2- breast cancer, triple negative breast cancer (TNBC) or HER2+ breast cancer.

172. A composition of genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs) produced by the method of any of claims 1-171.

173. A SOCS-1 targeting gRNA comprising a spacer sequence set forth in any one of SEQ ID NOS: 35-44.

174. The SOCS-1 targeting gRNA of claim 173, wherein the gRNA comprises a spacer sequence set forth in SEQ ID NO: 35.

175. The SOCS-1 targeting gRNA of claim 173, wherein the gRNA comprises a spacer sequence set forth in SEQ ID NO: 36.

176. The SOCS-1 targeting gRNA of claim 173, wherein the gRNA comprises a spacer sequence set forth in SEQ ID NO: 42.

177. The SOCS-1 targeting gRNA of claim 173, wherein the gRNA comprises a spacer sequence set forth in SEQ ID NO: 43.

178. The SOCS-1 targeting gRNA of claim 173, wherein the gRNA comprises a spacer sequence set forth in SEQ ID NO: 44.

179. A CISH-targeting gRNA comprising a spacer sequence set forth in any one of SEQ ID NOS: 23-29.

180. The CISH-targeting gRNA of claim 179, wherein the gRNA comprises a spacer sequence set forth in any one of SEQ ID NOS: 23-27.

181. The CISH-targeting gRNA of claim 179 or claim 180 wherein the gRNA comprises a spacer sequence set forth in SEQ ID NO: 27.

182. A PD-1 -targeting gRNA comprising a spacer sequence set forth in any one of SEQ ID NOS: 30-34.

183. A PD-1 -targeting gRNA of claim 182, wherein the gRNA comprises a spacer sequence set forth in SEQ ID NO:32.

184. The gRNAof any of claims 173-183, wherein the gRNA further comprises a scaffold sequence (tracR RNA) for complexing with a Cas protein.

185. A CRISPR-Cas combination comprising a guide RNA (gRNA) of any of claims 173-183 and a Cas nuclease.

186. The CRISPR-Cas combination of claim 185, wherein the Cas nuclease is a Cas9.

187. The CRISPR-Cas combination of claim 186, wherein the Cas9 is a streptococcus pyogenes Cas9 (spCas9).

188. The CRISPR-Cas combination of claim 185, wherein the Cas nuclease is Mad7.

189. The CRISPR-Cas combination of any of claims 185-188, wherein the gene editing agent is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease.

190. A vector comprising the CRISPR-Cas combination of any of claims 185-189.

191. The vector of claim 190, wherein the vector is a lipid nanoparticle or is a viral vector.

192. A composition comprising a population of genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), wherein at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the total cells or total T cells in the composition comprise a genetic disruption at a target site in a SOCS1 target gene.

193. A composition comprising a population of genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), wherein at least at or about 70%, 75%, 80%, 85%, 90%,or 95% of the total cells or total T cells in the composition comprise a genetic disruption at a target site in a C1SH target gene.

194. A composition comprising a population of genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs), wherein at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the total cells or total T cells in the composition comprise a genetic disruption in a target site in a PDCD1 (PD-1) target gene.

195. The composition of any of claims 192-194, wherein the genetic disruption is an indel.

196. The composition of any of claims 192-195, wherein the genetic disruption inactivates all alleles of the target gene.

197. The composition of any of claims 192-196, wherein the genetic disruption knock out expression of the target gene, optionally wherein expression is genomic expression or is protein expression on the cell surface as determined by flow cytometry.

198. The composition of claim 136 or any of claims 192-197, wherein at least 90% of cells in the composition are CD3+ T cells and less than about 5% of the population are T regulatory cells.

199. The composition of claim 96 or any of claims 192-198, wherein the population of genetically modified tumor-reactive tumor infiltrating lymphocytes (TILs) are an oligoclonal population.

200. The composition of claim 199, wherein up to 40 clones make up at least 40% of the TCR frequency in the population.

201. The composition of any of claims 172 and 192-200, wherein the composition is for treatment of a patient’s tumor.

202. The composition of claim 201, wherein the tumor is a kidney, lung, colorectal cancer (CRC) tumor, a melanoma tumor, a non-small cell lung cancer (NSCLC) tumor, or an ovarian cancer tumor.

203. The composition of claim 201 or claim 202, wherein the tumor is from a human subject.

204. The composition of claim 203, wherein the composition is for autologous adoptive therapy to the human subject.

205. The composition of any of claims 192-204, that is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

206. The composition of any of claims 192-205, comprising a cryoprotectant.

207. The composition of any of claims 201-206, wherein the composition is a liquid composition.

208. The composition of claim 207, wherein the composition had been frozen and thawed.

209. The composition of any of claims 201-208, wherein the volume of the composition is between 1 mL and 500 mL.

210. The composition of any of claims 201-209, wherein the composition is frozen.

211. A method of treating a solid tumor malignancy in a subject, the method comprising administering a T lymphocyte infiltrating (TIL) composition of any of claims 172 and 192-210 to the subject.

212. The method of claim 211, wherein the tumor is a tumor of an epithelial cancer.

213. The method of claim 211 or claim 212, wherein the tumor is a tumor of a lung (e.g., lung squamous, lung adenocarcinoma, lung small cell cancer), kidney, melanoma, bladder cancer, esophageal cancer, colorectal cancer (CRC), cervical cancer, head and neck cancer, stomach cancer or uterine cancer.

214. The method of any of claims 211-213, wherein the tumor is a tumor of a nonsmall cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma cancer, endometrial cancer, optionally wherein the breast cancer is HR+ / Her2- breast cancer, triple negative breast cancer (TNBC) or HER2+ breast cancer.

215. The method of claims 211-214, wherein the solid tumor malignancy is selected from the group comprising: breast cancer, colorectal carcinoma (CRC), uveal melanoma, cutaneous carcinoma, non- small cell lung cancer, head and neck squamous cell carcinoma, or any advanced solid tumor.

216. The method of any of claims 211-215, wherein the method further comprises administration of a lymphodepleting therapy.

217. The method of claim 216, wherein the lymphodepleting therapy is or comprises cyclophosphamide, ifosfamide, altretamine, busulfan, carboplatin, carmustine, cisplatin, dacarbazine, lomustine, melphalan, temozolomide, trabectedin, sodium 2-mercaptoethane sulfonate (Mensa), fludarabine, floxuridine, or any combination thereof.

218. The method of claim 216 or claim 217, wherein the lymphodepleting therapy is or comprises cyclophosphamide, sodium 2-mercaptoethane sulfonate (Mensa), fludarabine, or a combination thereof.

219. The method of any of claims 216-218, wherein the lymphodepleting therapy comprises administration of cyclophosphamide from about 30 mg / kg to about 200 mg / kg, optionally wherein the cyclophosphamide is administered at or about 60 mg / kg, optionally wherein the cyclophosphamide is administered within about 5 to 9 days prior to administeringthe TIL composition, more optionally wherein the cyclophosphamide is administered about 7 days prior to administering the TIL composition (Day -7).

220. The method of any of claims 216-218, wherein the lymphodepleting therapy comprises administration of Mensa from about 5 mg / kg / day to 25 mg / kg / day, optionally wherein the Mensa is administered at or about 15 mg / kg / day, optionally wherein the Mensa is administered within about 4 to 8 days prior to administering the TIL composition, more optionally wherein the Mensa is administered about 6 days prior to administering the TIL composition (Day -6) .

221. The method of any of claims 216-220, wherein the lymphodepleting therapy comprises administration of fludarabine from about 20-40 mg / m2, optionally at or about 25 mg / m2daily, optionally wherein the fludarabine is administered for three consecutive days, optionally wherein the fludarabine is administered 3 to 5 days prior to administering the TIL composition (Day -5 to Day -3).

222. The method of any of claims 216-221, wherein the administration of the lymphodepleting therapy is completed within about 2 to 7 days prior to initiation of the administration of the TIL composition, optionally 2 to 5 days prior to initiation of the administration of the TIL composition.

223. The method of any of claims 211-222, further comprising administering recombinant IL-2 to the subject.

224. The method of claim 223, wherein the initiation of administration of the recombinant IL-2 is 2 hour to 24 hours after administration of the TIL composition, optionally wherein initiation of administration of the recombinant IL-2 is 2-3 hours after administration of the TIL composition.

225. The method of claim 223 or claim 224, wherein each dose of the recombinant IL- 2 is from 600,000 lU / kg and 1,000,000 lU / kg, optionally administered 3 to 4 times a day for up to 3 days.

226. The method of any of claims 223-225, wherein the recombinant IL-2 isAldesleukin.

227. The method of any of claims 211-222, wherein the TIL composition is administered without administering recombinant IL-2 to the subject.

228. The method of claim 226 wherein the TIL composition comprises a population of genetically modified TIL with a genetic disruption of the SOCS1 locus.

229. The method of any of claims 1-2, 4-10, 12-20, and 22-171, wherein the target gene is selected from the group consisting of SOCS1, CISH, PDCD1 (PD-1), TIGIT, RC3H1 (Roquin), CMIP (gene encoding C-Maf Inducing Protein), CD39, LAG3, TIM3, MAPK14, and RASA2 (gene encoding RAS p21 protein activator 2).

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